Hepatitis B viral variants with reduced susceptibility to nucleoside analogs and uses thereof

ABSTRACT

The present invention relates generally to viral variants exhibiting reduced sensitivity to particular agents and/or reduced interactivity with immunological reagents. More particularly, the present invention is directed to hepatitis B virus (HBV) variants exhibiting complete or partial resistance to nucleoside or nucleotide analogs and/or reduced interactivity with antibodies to viral surface components including reduced sensitivity to these antibodies. The present invention further contemplates assays for detecting such viral variants, which assays are useful in monitoring anti-viral therapeutic regimens and in developing new or modified vaccines directed against viral agents and in particular HBV variants. The present invention also contemplates the use of the viral variants to screen for and/or develop or design agents capable of inhibiting infection, replication and/or release of the virus.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. patent application Ser. No.11/860,727, filed Sep. 25, 2007, now U.S. Pat. No. 7,745,130 which is acontinuation of U.S. patent application Ser. No. 11/166,004 filed Jun.24, 2005, now U.S. Pat. No. 7,384,747 which is a continuation of U.S.patent application Ser. No. 10/963,333 filed Oct. 12, 2004, nowabandoned which is a continuation of International Patent ApplicationNo. PCT/AU03/00432, filed Apr. 11, 2003 and published in English on Oct.23, 2003, as International Patent Publication No. WO03/087351, whichclaims priority to Australian Patent Application Nos. PS 1710, filedApr. 12, 2002, and PS 3224, filed Jun. 26, 2002, each of which is byreference incorporated herein in its entirety and to each of whichpriority is claimed.

SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listingsubmitted herewith via EFS on Jun. 1, 2010. Pursuant to 37 C.F.R.§1.52(e)(5), the Sequence Listing text file, identified as0718380158SeqList.TXT, is 99,218 bytes and was created on Jun. 1, 2010.The Sequence Listing, electronically filed herewith, does not extendbeyond the scope of the specification and thus does not contain newmatter.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to viral variants exhibitingreduced sensitivity to particular agents and/or reduced interactivitywith immunological reagents. More particularly, the present invention isdirected to hepatitis B virus (HBV) variants exhibiting complete orpartial resistance to nucleoside or nucleotide analogs and/or reducedinteractivity with antibodies to viral surface components includingreduced sensitivity to these antibodies. The present invention furthercontemplates assays for detecting such viral variants, which assays areuseful in monitoring anti-viral therapeutic regimens and in developingnew or modified vaccines directed against viral agents and in particularHBV variants. The present invention also contemplates the use of theviral variants to screen for and/or develop or design agents capable ofinhibiting infection, replication and/or release of the virus.

2. Description of the Prior Art

Bibliographic details of the publications referred to in thisspecification are also collected at the end of the description.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in any country.

Hepatitis B virus (HBV) can cause debilitating disease conditions andcan lead to acute liver failure. HBV is a DNA virus which replicates viaan RNA intermediate and utilizes reverse transcription in itsreplication strategy (Summers and Mason, Cell 29: 403-415, 1982). TheHBV genome is of a complex nature having a partially double-stranded DNAstructure with overlapping open reading frames encoding surface, core,polymerase and X genes. The complex nature of the HBV genome isrepresented in FIG. 1. The polymerase consists of four functionalregions, the terminal protein (TP), spacer, reverse transcriptase (rt)and ribonuclease (RNAse).

The polymerase gene of RSV overlaps the envelope gene, mutations in thecatalytic domain of the polymerase gene can also affect the nucleotideand the deduced amino acid sequence of the envelope protein and viceversa. In particular, the genetic sequence for the neutralization domainof HBV known as the ‘a’ determinant, which is found within the HBsAg andlocated between amino acids 99 and 169, actually overlaps the majorcatalytic regions of the viral polymerase protein and in particulardomains A and B.

The presence of an HBV DNA polymerase has led to the proposition thatnucleoside or nucleotide analogs could act as effective anti-viralagents. Examples of nucleoside analogs currently being tested arepenciclovir and its oral form (FCV) [Vere Hodge, Antiviral ChemChemother 4: 67-84, 1993; Boyd et al., Antiviral Chem Chemother. 32:358-363, 1987; Kruger et al., Hepatology 22: 219A, 1994; Main et al., J.Viral Hepatitis 3: 211-215, 1996],Lamivudine[(−)-β-2′-deoxy-3′-thiacytidine]; (3TC or LMV) [Severini etal., Antimicrobial Agents Chemother. 39: 430-435, 1995; Dienstag et al.,New England J Med 333: 1657-1661, 1995]. New nucleoside or nucleotideanalogs which have already progressed to clinical trials include thepyrimidines Emtricitabine,((−)-(β-L-2′-3′-dideoxy-5-fluoro-3′-thiacydidine; FTC), the 5-fluoroderivative of 3TC, and Clevudine(1-(2-fluoro-5-methyl-β-L-arabino-furanosyl) uracil; L-FMAU), athymidine analog: Like 3TC, these are pyrimidine derivatives with anunnatural “L”-configuration. Several purine derivatives have alsoprogressed to clinical trials; they include Entecavir (BMS-200, 475;ETV), a carbocyclic deoxyguanosine analog, diaminopurine dioxolane(DAPD), an oral pro-drug for dioxolane guanine ((−)-β-D-2-aminopurinedioxolane; DXG) and Adefovir dipivoxil, an oral prodrug for the acyclicdeoxyadenosine monophosphate nucleoside analog Adefovir(9-[phosphonyl-methoxyethyl]adenine; PMEA). Other drugs in pre-clincialand clinical trials include FLG [Medivir], ACH-126,443 (L-d4C)[Archillion Pharmaceuticals], ICN 2001-3 (ICN) and Racivir (RCV)[Pharmassett]. Whilst these agents are highly effective in inhibitingHBV DNA synthesis, there is the potential for resistant mutants of HBVto emerge during long term antiviral chemotherapy. In patients onprolonged LMV therapy, key resistance mutations are selected in the rtdomain within the polymerase at rtM204I/V+/−rtL180M as well as othermutations. The nomenclature used for the polymerase mutations is inaccordance with that proposed by Stuyver et al., 2001, supra. LMV is anucleoside analog that has been approved for use against chronic HBVinfection. LMV is a particularly potent inhibitor of HBV replication andreduces HBV DNA titres in the sera of chronically infected patientsafter orthotopic liver transplantation (OLT) by inhibiting viral DNAsynthesis. LMV monotherapy seems unlikely to be able to control HBVreplication in the longer term. This is because emergence ofLMV-resistant strains of HBV seems almost inevitable during monotherapy.

Adefovir dipivoxil (ADV: formerly, bis-pom PMEA) is an orally availableprodrug of the acyclic deoxyadenosine monophosphate analog adefovir(formerly, PMEA) (FIG. 2). ADV is also a potent inhibitor of HBVreplication and has recently been given FDA approval for use againstchronic HBV infection. Adefovir dipivoxil differs from other agents inthis class in that it is a nucleotide (vs. nucleoside) analog and assuch bypasses the first phosphorylation reaction during drug activation.This step is often rate-limiting. Adefovir dipivoxil has demonstratedclinical activity against both wild-type and lamivudine-resistantstrains of HBV and is currently in phase III clinical Testing (Gilson etal, J Viral Hepat 6: 387-395, 1999; Perrino et al., Hepatology 32:129-134, 2000; Peters et al., Transplantation 68: 1912-1914, 1999;Benhamou et al., Lancet 358: 718-723, 2001). During phase II studies a30 mg daily dose of adefovir dipivoxil resulted in a mean 4 log₁₀decrease in viremia over 12 weeks (Heathcote et al., Hepatology 28:A620, 1998).

ADV is a substituted acyclic nucleoside phosphonate. This class ofcompounds also includes tenofovir disoproxil fumarate (also referred toas tenofovir DF, or tenofovir, or (TFV) or9-R-(2-phosphonomethoxypropyl)adenine (PMPA) and is marketed as Vireadby Gilead sciences).

TFV has antiviral activity against both HBV and HIV (Ying et al., JViral Hepat. 7(2): 161-165, 2000; Ying et al., J. Viral Hepat. 7(1):79-83, 2000; Suo et al., J Biol Chem. 273(42): 27250-27258. 1998).

FTC has activity against HBV and HIV (Frick et al., Antimicrob AgentsChemother 37: 2285-2292, 1993).

Nucleoside or nucleotide analog therapy may be administered asmonotherapy or combination therapy where two or more nucleoside ornucleotide analogs may be administered. The nucleoside or nucleotideanalogs may also be administered in combination with other antiviralagents such as interferon or hepatitis B immunoglobulin (HBIG).

There is a need to monitor for the emergence ofnucleoside/nucleotide-analog- or antibody-resistant strains of HBV andto develop diagnostic protocols to detect these resistant viruses and/orto use them to screen for and/or develop or design agents havingproperties making them useful as anti-viral agents. Defective forms ofthese resistant strains or antigenic components therefrom are alsoproposed to be useful in the development of therapeutic vaccinecompositions as are antibodies directed to viral surface components.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2),etc. A summary of the sequence identifiers is provided in Table 1. Asequence listing is provided after the claims.

Specific mutations in an amino acid sequence are represented herein as“Xaa₁nXaa₂” where Xaa₁ is the original amino acid residue beforemutation, n is the residue number and Xaa₂ is the mutant amino acid. Theabbreviation “Xaa” may be the three letter or single letter (i.e. “X”)code. An “rt” before “Xaa₁nXaa₂” means “reverse transcriptase”. An “s”means an envelope gene. The amino acid residues for HBV DNA polymeraseare numbered with the residue methionine in the motif Tyr Met Asp Asp(YMDD) being residue number 204 (Stuyver et al., Hepatology 33: 751-757,2001). The amino acid residues for hepatitis B virus surface antigen arenumber according to Norder et al. (J. Gen. Virol. 74: 341-1348, 1993).Both single and three letter abbreviations are used to define amino acidresidues and these are summarized in Table 2.

In accordance with the present invention, the selection of HBV variantsis identified in patients (Patient A, C and D) with chronic HBVinfection treated with ADV and liver transplant patients (Patients B andE) treated with both ADV and LMV post-OLT or ADV post-transplant. HBVvariants from Patients F, G and H were also identified following similartreatments. Variants of HBV are identified during ADV or combination ADVand LMV treatment with mutations in the HBV DNA polymerase gene whichreduce the sensitivity of HBV to this nucleoside analog. Consequently,HBV rt variants are contemplated which are resistant to, or whichexhibit reduced sensitivity to, ADV, LMV, TFV, FTC, ADV and LMV, ADV andTFV, LMV and TFV, FTC and ADV, FTC and TFV, FTC and LMV, or ADV and LMVand TFV, or ADV and FTC and TFV, TFV and FTC and LMV, ADV and LMV andFTC, or ADV and FTC and LMV and TFV and/or optionally other nucleosideor nucleotide analogs or other anti-HBV agentsor combinations thereof.Corresponding mutations in the surface antigen also occur. Theidentification of these HBV variants is important for the development ofassays to monitor ADV, LMV, FTC and/or TFV resistance and/or resistanceto other nucleoside or nucleotide analogs or other anti-HBV agents orcombinations thereof and to screen for agents which are useful asalternative therapeutic agents.

Reference herein to “anti-HBV agents” includes nucleoside and nucleotideanalogs as well as immunological reagents (e.g. antibodies to HBVsurface components) and chemical, proteinaceous and nucleic acid agentswhich inhibit or otherwise interfere with viral replication,maintenance, infection, assembly or release.

The detection of such HBV variants is particularly important in themanagement of therapeutic protocols including the selection ofappropriate agents for treating HBV infection. The method of this aspectof the present invention is predicated in part on monitoring thedevelopment in a subject of an increased HBV load in the presence of anucleoside or nucleotide analog or other anti-HBV agents or combinationsthereof. The clinician is then able to modify an existing treatmentprotocol or select an appropriate treatment protocol accordingly.

Accordingly, one aspect of the present invention is directed to anisolated HBV variant comprising a nucleotide mutation in a gene encodinga DNA polymerase resulting in at least one amino acid addition,substitution and/or deletion to the DNA polymerase and which exhibitsdecreased sensitivity to ADV, LMV, TFV, or FTC, or ADV and LMV, ADV andTFV, LMV and TFV, FTC and ADV, FTC and TFV, FTC and LMV, or ADV and LMVand TFV, or ADV and FTC and TFV, TFV and FTC and LMV, ADV and LMV andFTC, or ADV and FTC and LMV and TFV and/or optionally other nucleosideor nucleotide analogs or other anti-HBV agents or combinations thereof.The variant HBV comprises a mutation in an overlapping open readingframe in its genome in a region defined by one or more of domains F andG and domain A through to E of HBV DNA polymerase.

Another aspect of the present invention provides an isolated HBV variantcomprising a nucleotide mutation in the S gene resulting in at least oneamino acid addition, substitution and/or deletion to the surface antigenand which exhibits decreased sensitivity to ADV, LMV, TFV, or FTC, orADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV, FTC and TFV, FTC andLMV, or ADV and LMV and TFV, or ADV and FTC and TFV, TFV and FTC andLMV, or ADV and FTC and LMV and TFV, ADV and LMV and FTC, and/oroptionally other nucleoside or nucleotide analogs or other anti-HBVagents or combinations thereof.

Useful mutants in the rt region include, in one embodiment, rtS21A,rtL122F, rtN124H, rtH126R, rtT28N, rtP130Q, rtD131N and rtY135C; inanother embodiment, rt/N/S/T/I/V53D, rtY126Q, rtL180M, rtS202G, rtI204Vand rtI235UM; in a further embodiment, rtN53D, rtY54H, rtS57P, rtL91I,rtS116P, rtF122L, rtY124H, rtV134D, rtY141Y/F, rtL145M, rtF151F/Y,rtA181T, rtK212R, rtL217R, rtS219A, rtN236T and rtN238D; in yet anotherembodiment, rtS78T, rtV84M, rtY126C, rtV191I, rtM204I and rtV214A; andin yet another embodiment, rtH90D and rtL/F108L; and in still a furtherembodiment, rtL157L/M, rtA181V and rtV207I and in yet a furtherembodiment, rtL80V, rtP109S, rtI163V, rtL229M and rtN/H/A/S/Q238K; andin another embodiment, rtS78S/T, rtN118N/S, rtN139N/K, rtV142E,rtA181A/T, rtI204M, rtQ/P/S/Stop215Q, rtE218K/E and rtN238N/H or acombination thereof or an equivalent mutation.

Other HBV variants are also contemplated with mutations in rt at rtK32,rtN33, rtP34, rtH35 and rtT37 (these are upstream of the F domain of theDNA polymerase), rtP59, rtK60, rtF61, rtA62 and rtV63 (these are locatedbetween the F and A domains), rtD83, rtV84, rtS85, rtA86, rtY89, rtH90and rtI/L91 (these are located within the A domain and the regionimmediately prior to and following), rtP177, rtF178, rtL179, rtL180,rtA181, rtQ182, rtF183 and rtT184 (these are located in the B domain),rtM204 and rtY203 (these are located in the C domain), rt235, rt236,rt237, rt238 and rt239 (these are located in the D domain) and rt247,rt248, rt249, rt250 and rt251 (these are located in the E domain) or acombination thereof or an equivalent mutation.

Useful mutants are provided below (see also Tables 16 and 17):

K32M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;N33D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/deletion;P34S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;H35I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;T37W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/deletion;P59S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;K60M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;F61P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/deletion;A62R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/deletion;V63A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;D83C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/deletion;V84A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;S85T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/deletion;A86R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/YV/deletion;Y89V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/deletion;H90I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;I/L91K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/deletion;P177S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;F178P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;L179K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;L180K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;A181R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;Q183E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;F183P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;T184W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;Y203V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/ deletion;M204F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;L235K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N236D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;T237W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;P237S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;N238D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H238I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;A238R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;S239T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/ deletion;Q238E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;K239M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/ deletion;L247K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N248D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H248I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;F249P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;M250F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;G251H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/ deletion; andV251A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/ deletion.

Reference above to “deletion” means that the first mentioned amino acidbefore the residue number has been deleted.

Useful mutations in the S gene include, in one embodiment, sP120T,sM125T and sT127A; in another embodiment, T118R, sM133T, sF134V sI195M,sS207R and sY225Y/C; in a further embodiment, sS126T, sM133L/M,sS143S/T, sD144A sG145A and sW172Stop; in yet a further embodiment,sN40S, sC69 Stop, sM75I, sL88P, sT118A, sW182stop, sW196L, sY206H andsY225F; and in yet another embodiment, sI81M and sP214Q; and in stillanother embodiment, sF83S, sL173F and sW199L; and in still yet anotherembodiment, sI126T, sK160R, sS174N, sA184V, sW196L, sS210N, sF/C220L andsY221C; and in yet another embodiment, sC69Stop/C, sC76Y sI110V/I,sY134N, sW172Stop/W, sW196Stop and sS207R or a combination thereof or anequivalent mutation.

The present invention further contemplates a method for determining thepotential for an HBV to exhibit reduced sensitivity to ADV, LMV, TFV, orFTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV, FTC and TFV,FTC and LMV, or ADV and LMV and TFV, or ADV and FTC and TFV, TFV and FTCand LMV, ADV and LMV and FTC, or ADV and FTC and LMV and TFV and/oroptionally other nucleoside or nucleotide analogs or other anti-HBVagents or combination thereof by isolating DNA or corresponding mRNAfrom the HBV and screening for a mutation in the nucleotide sequenceencoding HBV DNA polymerase resulting in at least one amino acidsubstitution, deletion and/or addition in any one or more of domains Fand G and domains A through to E or a region proximal thereto of the DNApolymerase and associated with resistance or decreased sensitivity toADV, LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC andADV, FTC and TFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTCand TFV, TFV and FTC and LMV, ADV and LMV and FTC, or ADV and FTC andLMV and TFV and/or optionally other nucleoside or nucleotide analogs orother anti-HBV agents or combination thereof. The presence of such amutation is an indication of the likelihood of resistance to ADV, LMV,TFV, or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV, FTCand TFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTC and TFV,TFV and FTC and LMV, ADV and LMV and FTC, or ADV and FTC and LMV and TFVand/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combination thereof.

The present invention also provides a composition comprising a variantHBV resistant to ADV, LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMVand TFV, FTC and ADV, FTC and TFV, FTC and LMV, or ADV and LMV and TFV,or ADV and FTC and TFV, TFV and FTC and LMV, or ADV and FTC and LMV andTFV, ADV and LMV and FTC, and/or optionally other nucleoside ornucleotide analogs or other anti-HBV agents or combination thereof or anHBV surface antigen from the variant HBV or a recombinant or derivativeform thereof or its chemical equivalent and one or more pharmaceuticallyacceptable carriers and/or diluents.

Yet another aspect of the present invention provides a use of theaforementioned composition or a variant HBV comprising a nucleotidemutation in a gene encoding a DNA polymerase resulting in at least oneamino acid addition, substitution and/or deletion to the DNA polymeraseand a decreased sensitivity to ADV, LMV, TFV, or FTC, or ADV and LMV,ADV and TFV, LMV and TFV, FTC and ADV, FTC and TFV, FTC and LMV, or ADVand LMV and TFV, or ADV and FTC and TFV, TFV and FTC and LMV, ADV andLMV and FTC, or ADV and FTC and LMV and TFV and/or optionally othernucleoside or nucleotide analogs or other anti-HBV agents or combinationthereof in the manufacture of a medicament for the treatment and/orprophylaxis of hepatitis B virus infection.

The present invention also contemplates a method for determining whetheran HBV strain exhibits reduced sensitivity to a nucleoside or nucleotideanalog or other anti-HBV agents or by isolating DNA or correspondingmRNA from the HBV and screening for a mutation in the nucleotidesequence encoding the DNA polymerase wherein the presence of thefollowing mutations in the rt region: in one embodiment, rtS21A,rtL122F, rtN124H, rtH126R, rtT28N, rtP130Q, rtD131N and rtY135C; inanother embodiment, rt/N/S/T/1/V53D, rtY126Q, rtL180M, rtS202G, rtI204Vand rtI235I/M; in a further embodiment, rtN53D, rtY54H, rtS57P, rtL91I,rtS116P, rtF122L, rtY124H, rtV134D, rtY141Y/F, rtL145M, rtF151F/Y,rtA181T, rtL212R, rtL217R, rtS219A, rtN236T and rtN238D; in yet anotherembodiment, rtS78T, rtV84M, rtY126C, rtV191I, rtM204I and rtV214A; instill another embodiment, rtH90D and rtL/F108L, in even yet anotherembodiment, rtL157L/M, rtA181V and rtV207I; in still yet anotherembodiment, rtL80V, rtP109S, rtI163V, rtL229M and rtN/H/A/S/Q238K; inanother embodiment, rtS78S/T, rtN118N/S, rtN139N/K, rtV142E, rtA181A/T,rtI204M, rtQ/P/S/Stop215Q, rtE218K/E and rtN238N/H; in a furtherembodiment, rtK32, rtN33, rtP34, rtH35 and rtT37; in yet anotherembodiment, rtP59, rtK60, rtF61, rtA62 and rtV63; in still anotherembodiment, rtD83, rtV84, rtS85, rtA86, rtY89, rtH90 and rtI/L91; ineven yet another embodiment, rtP177, rtF178, rtL179, rtL180, rtA181,rtQ182, rtF183 and rtT184; in still yet another embodiment, rtM204 andrtY203; in another embodiment, rt235, rt236, rt237, rt238 and rt239; ina further embodiment, rt247, rt248, rt249, rt250 and rt251 orcombinations thereof or an equivalent one or more other mutation isindicative of a variant which exhibits a decreased sensitivity to ADV,LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV,FTC and TFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTC andTFV, TFV and FTC and LMV, ADV and LMV and FTC, or ADV and FTC and LMVand TFV and/or optionally other nucleoside or nucleotide analogs orother anti-HBV agents or combination thereof.

Still a further methodology comprises screening for a mutation in thenucleotide sequence encoding the envelope genes (s) wherein the presenceof the following mutations in the S gene: in one embodiment, sP120T,sM125T and sT127A; in another embodiment, sT118R, sM133T, SF134V,sI195M, sS207R and sY225Y/C; in a further embodiment, sS126T, sM133L/M,sS143S/T, sD144A, sG145A and sW172Stop in yet another embodiment, sN40S,sC69Stop, sM75I, sL88P, sT118A, sW182Stop, sW196L, sY206H and sY225F; instill yet another embodiment, s181M and sP214Q; in another embodiment,sF83S, sL173F and sW199L; in a further aspect, sI126T, sK160R, sS174N,sA184V, sW196L, sS210N, sF/C220L and sY221C; in a further embodiment,sC69Stop/C, sC76Y, sI110V/I, sY134N, sW172Stop/W, sW196Stop and sS207Ror combinations thereof or an equivalent one or more other mutation isindicative of a variant which exhibits a decreased sensitivity to ADV,LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV,FTC and TFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTC andTFV, TFV and FTC and LMV, ADV and LMV and FTC, or ADV and FTC and LMVand TFV, and/or optionally other nucleoside or nucleotide analogs orother anti-HBV agents or combination thereof.

Preferably, the variants are in an isolated form such that they haveundergone at least one purification step away from naturally occurringbody fluid. Alternatively, the variants may be maintained in isolatedbody fluid or may be in DNA form. The present invention alsocontemplates infectious molecular clones comprising the genome or partsthereof from a variant HBV. The detection of HBV or its components incells, cell lysates, cultured supernatant fluid and bodily fluid may beby any convenient means including any nucleic acid-based detectionmeans, for example, by nucleic acid hybridization techniques or via oneor more polymerase chain reactions (PCBs). The term “bodily fluid”includes any fluid derived from the blood, lymph, tissue or organsystems including serum, whole blood, biopsy and biopsy fluid, organexplants and organ suspension such as liver suspensions.

Another aspect of the present invention is directed to a variant HBVcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion or atruncation compared to a surface antigen from a reference or wild typeHBV and wherein an antibody generated to the reference or wild typesurface antigen exhibits an altered immunological profile relative tothe HBV variant. One altered profile includes a reduced capacity forneutralizing the HBV. More particularly, the surface antigen of thevariant HBV exhibits an altered immunological profile compared to apre-treatment HBV where the variant HBV is selected for by a nucleosideor nucleotide analog or other anti-HBV agents of the HBV DNA polymerase.The variant HBV of this aspect of the invention may also comprise anucleotide sequence comprising a single or multiple nucleotidesubstitution, addition and/or deletion compared to a pre-treatment HBV.

The present invention extends to an isolated HBsAg or a recombinant formthereof or derivative or chemical equivalent thereof corresponding tothe variant HBV. Generally, the HBsAg or its recombinant or derivativeform or its chemical equivalent comprises an amino acid sequence with asingle or multiple amino acid substitution, addition and/or deletion ora truncation compared to an HBsAg from a reference HBV and wherein anantibody directed to a reference HBV exhibits an altered immunologicalprofile to an HBV carrying said variant HBsAg. In one embodiment, thealtered immunological profile comprises a reduction in the ability toneutralize the variant HBV.

Another aspect of the present invention contemplates a method fordetecting an agent which exhibits inhibitory activity to an HBV bygenerating a genetic construct comprising a replicationcompetent-effective amount of the genome from the HBV contained in aplasmid vector and then transfecting said cells with said construct,contacting the cells, before, during and/or after transfection, with theagent to be tested, culturing the cells for a time and under conditionssufficient for the HBV to replicate, express genetic sequences and/orassemble and/or release virus or virus-like particles if resistant tosaid agents; and the subjecting the cells, cell lysates or culturesupernatant fluid to viral- or viral-component-detection means todetermine whether or not the virus has replicated, expressed geneticmaterial and/or assembled and/or been released in the presence of theagent. In a preferred embodiment, the plasmid vector in a baculovirusvector and the method comprises generating a genetic constructcomprising a replication competent-effective amount of the genome fromthe HBV contained in or fused to an amount of a baculovirus genomeeffective to infect cells and then infecting said cells with saidconstruct, contacting the cells, before, during and/or after infection,with the agent to be tested, culturing the cells for a time and underconditions sufficient for the HBV to replicate, express geneticsequences and/or assemble and/or release virus or virus-like particlesif resistant to said agent and then subjecting the cells, cell lysatesor culture supernatant fluid to viral- or viral-component-detectionmeans to determine whether or not the virus has replicated, expressedgenetic material and/or assembled and/or been released in the presenceof the agent.

In connection with these methods, the plasmid vector may include genesencoding part or all of other viral vectors such as baculovirus vectorsor adenovirus vectors (see Ren and Nassal, J. Virol. 75(3): 1104-1116,2001).

In an alternative embodiment, the method comprises generating acontinuous cell line comprising an infectious copy of the genome of theHBV in a replication competent effective amount such that saidinfectious HBV genome is stably integrated into said continuous cellline such as but not limited to the 2.2.15 or AD cell line, contactingthe cells with the agent to be tested, culturing the cells for a timeand under conditions sufficient for the HBV to replicate, expressgenetic sequences and/or assemble and/or release virus or virus-likeparticles if resistant to the agent and then subjecting the cells, celllysates or culture supernatant fluid to viral- orviral-component-detection means to determine whether or not the virushas replicated, expressed genetic material and/or assembled and/or beenreleased in the presence of the agent.

In an alternative embodiment, the present invention also contemplates amethod for detecting an agent which exhibits inhibitory activity to anHBV polymerase in an in vitro polymerase assay. The HBV polymeraseactivity can be examined using established assays (Gaillard et al.,Antimicrob Agents Chemother. 46(4): 1005-1013, 2002; Xiong et al.,Hepatology. 28(6): 1669-73, 1998). The HBV polymerase may be a wild-typeor reference HBV polymerase or mutant HBV polymerase.

The identification of viral variants enables the production of vaccinescomprising particular recombinant viral components such as polymerasesor envelope genes PreS1, PreS2, S encoding for L, M, S proteins as wellas therapeutic vaccines comprising defective HBV variants. Rational drugdesign may also be employed to identify or generate therapeuticmolecules capable of interacting with a polymerase or envelope genesPreS1, PreS2, S encoding for L, M, S proteins or other component of theHBV. Such drugs may also have diagnostic potential. In addition,defective HBV variants may also be used as therapeutic compositions togenerate an immune response against the same, similar or homologousviruses. Alternatively, antibodies generated to the HBV variants orsurface components thereof may be used in passive immunization ofsubjects against infection by HBV variants or similar or homologousviruses. Furthermore, agents such as nucleoside or nucleotide analogs,RNAi or siRNA molecules, antisense or sense oligonucleotides, chemicalor proteinaceous molecules having an ability to down-regulate theactivity of a component of HBV and inhibit replication, maintenance,infection, assembly or release are contemplated by the presentinvention.

A summary of the abbreviations used throughout the subject specificationare provided in Table 3.

A summary of sequence identifiers used throughout the subjectspecification is provided in Table 1.

TABLE 1 Summary of sequence identifiers SEQUENCE ID NO: DESCRIPTION 1Formula I 2 Formula II 3 OS1 primer 4 TTA3 primer 5 JM primer 6 TTA4primer 7 OS2 primer 8 sense primer 9 antisense primer 10 internalregions primer 11 internal regions primer 12 PC1 forward primer 13 PC2reverse primer 14 HBV-specific molecular beacon primer 15 ILA 1 F, A-E(FIG. 4) 16 ILA 2 F, A-E (FIG. 4) 17 ILA 3 F, A-E (FIG. 4) 18 ILA 4 F,A-E (FIG. 4) 19 Pol Trans Pre 1 (FIG. 5) 20 Pol Trans 2 (FIG. 5) 21 PolTrans 3 (FIG. 5) 22 Pol Trans 4 (FIG. 5) 23 HBsAg Trans of Pre 1 (FIG.6) 24 HBsAg Trans of 2 (FIG. 6) 25 HBsAg Trans of 3 (FIG. 6) 26 HBsAgTrans of 4(FIG. 6) 27 S0 (FIG. 7) 28 S6 (FIG. 7) 29 S8 (FIG. 7) 30 S12(FIG. 7) 31 S15 (FIG. 7) 32 Pol Trans S0 (FIG. 8) 33 Pol Trans S6 (FIG.8) 34 Pol Trans S8 (FIG. 8) 35 Pol Trans S12 (FIG. 8) 36 Pol Trans S15(FIG. 8) 37 HBsAg Trans of S0 (FIG. 9) 38 HBsAg Trans of S6 (FIG. 9) 39HBsAg Trans of S8 (FIG. 9) 40 HBsAg Trans of S12 (FIG. 9) 41 HBsAg Transof S15 (FIG. 9) 42 Nucleotide sequence Patient C (FIG. 10) 43 POL Transof Patient C (FIG. 11) 44 HBsAg Trans of Patient C (FIG. 12) 45Nucleotide sequence of Patient D (FIG. 13) 46 Pol Trans of Patient D(FIG. 14) 47 HBsAg Trans of Patient D (FIG. 15) 48 Nucleotide sequenceof Patient E (FIG. 16) 49 Pol Trans of Patient E (FIG. 17) 50 HBsAgTrans of Patient E (FIG. 18) 51 Nucleotide sequence of Patient F (FIG.20) 52 Deduced sequence of DNA polymerase of Patient F (FIG. 21) 53HBsAg Trans of Patient F (FIG. 22) 54 Nucleotide sequence of Patient G(FIG. 23) 55 Deduced sequence of DNA polymerase of Patient G (FIG. 24)56 HBsAg Trans of Patient G (FIG. 25) 57 Nucleotide sequence of PatientH (FIG. 26) 58 Deduced sequence of DNA polymerase of Patient H (FIG. 27)59 HBsAg Trans of Patient H (FIG. 28)

TABLE 2 Single and three letter amino acid abbreviations Amino AcidThree-letter Abbreviation One-letter symbol Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln QGlutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile ILeucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F ProlinePro P Serine Ser S Threonine The T Tryptophan Trp W Tyrosine Tyr YValine Val V Any residue Xaa X

A list of abbreviations used throughout the subject specification areprovided in Table 3.

TABLE 3 Abbreviations ABBREVIATION DESCRIPTION 3TC (LMV);(−)-β-2′-deoxy-3′-thiacytidine ADV adefovir dipivoxil DAPD diaminopurinedioxalone DXG dioxolane guanine ETV entecavir FAM famciclovir FCVfamciclovir FTC emtricitabine HBIG hepatitis B immunoglobulin HBsAghepatitis B surface antigen HBV hepatitis B virus LMV lamividuine PMEA9-[phosphonyl-methoxyethyl]-adenine; adefovir PMPA9-R-(2-phosphonomethoxypropyl)adenine RNase ribonuclease rt (“rt” beforereverse transcriptase “Xaa₁nXaa₂” means reverse transcriptase) s (asused in a mutation, envelope gene e.g. sF134V) TFV tenofovir disoproxilfumarate YMDD Tyr Met Asp Asp-a motif in the polymerase protein; wherethe Met residue is designated residue number 204 of the reversetranscriptase

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatic representation showing the partially doublestranded DNA HBV genome showing the overlapping open reading framesencoding surface (S), core (C), polymerase (P) and X gene.

FIG. 2 is a diagrammatic representation of the chemical structure ofADV.

FIG. 3 is a diagrammatic representation of a computer system fordetermining the potency value (P_(A)) of a variant HBV.

FIG. 4 is a representation showing comparison of the HBV nucleotidesequence encoding the catalytic region of the polymerase gene insequential samples from Patient A during ADV treatment.

FIG. 5 is a representation showing comparison of the deduced amino acidsequence of the catalytic region of the polymerase gene in sequentialsamples from Patient A during ADV therapy.

FIG. 6 is a representation showing comparison of the deduced amino acidsequence of the envelope gene in sequential samples from Patient Aduring ADV therapy.

FIG. 7 is a representation showing comparison of the HBV nucleotidesequence encoding the catalytic region of the polymerase gene insequential samples from Patient B during ADV and LMV treatment.

FIG. 8 is a representation showing comparison of the deduced amino acidsequence of the catalytic region of the polymerase gene in sequentialsamples from Patient B during ADV and LMV therapy.

FIG. 9 is a representation showing comparison of the deduced amino acidsequence of the envelope gene in sequential samples from Patient Bduring ADV and LMV therapy.

FIG. 10 is a representation showing comparison of the HBV nucleotidesequence encoding the catalytic region of the polymerase gene insequential samples from Patient C during ADV treatment.

FIG. 11 is a representation showing comparison of the deduced amino acidsequence of the catalytic region of the polymerase gene in sequentialsamples from Patient C during ADV therapy.

FIG. 12 is a representation showing comparison of the deduced amino acidsequence of the envelope gene in sequential samples from Patient Cduring ADV therapy.

FIG. 13 is a representation showing comparison of the HBV nucleotidesequence encoding the catalytic region of the polymerase gene insequential samples from Patient D during ADV treatment.

FIG. 14 is a representation showing comparison of the deduced amino acidsequence of the catalytic region of the polymerase gene in sequentialsamples from Patient D during ADV therapy.

FIG. 15 is a representation showing comparison of the deduced amino acidsequence of the envelope gene in sequential samples from Patient Dduring ADV therapy.

FIG. 16 is a representation showing comparison of the HBV nucleotidesequence encoding the catalytic region of the polymerase gene insequential samples from Patient E during ADV treatment.

FIG. 17 is a representation showing comparison of the deduced amino acidsequence of the catalytic region of the polymerase gene in sequentialsamples from Patient E during ADV therapy.

FIG. 18 is a representation showing comparison of the deduced amino acidsequence of the envelope gene in sequential samples from Patient Eduring ADV therapy.

FIG. 19 is a diagrammatic representation of a system used to carry outthe instructions encoded by the storage medium.

FIG. 20 is a representation showing the nucleotide sequence ofenvelope/rt region of an HBV isolated from Patient F having ADV therapy.

FIG. 21 is a representation showing the deduced amino acid sequence ofDNA polymerase encoded by the nucleotide sequence shown in FIG. 20.

FIG. 22 is a representation showing the deduced amino acid sequence ofHBsAg encoded by the nucleotide sequence shown in FIG. 20.

FIG. 23 is a representation showing the nucleotide sequence ofenvelope/rt region of an HBV isolated from Patient G having ADV therapy.

FIG. 24 is a representation showing the deduced amino acid sequence ofDNA polymerase encoded by the nucleotide sequence shown in FIG. 23.

FIG. 25 is a representation showing the deduced amino acid sequence ofHBsAg encoded by the nucleotide sequence shown in FIG. 23.

FIG. 26 is a representation showing the nucleotide sequence ofenvelope/rt region of an HBV isolated from Patient H having ADV therapy.

FIG. 27 is a representation showing the deduced amino acid sequence ofDNA polymerase encoded by the nucleotide sequence shown in FIG. 26.

FIG. 28 is a representation showing the deduced amino acid sequence ofHBsAg encoded by the nucleotide sequence shown in FIG. 26.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated in part on the identification andisolation of nucleoside or nucleotide analog-resistant variants of HBVfollowing treatment of patients with either ADV or LMV or moreparticularly ADV and LMV, or optionally other nucleoside analogs ornucleotide analogs or other anti-HBV agents such as TFV or FTC. Inparticular, ADV or ADV and LMV treated patients gave rise to variants ofHBV exhibiting decreased or reduced sensitivity to ADV, LMV, TFV, orFTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV, FTC and TFV,FTC and LMV, or ADV and LMV and TFV, or ADV and FTC and TFV, TFV and FTCand LMV, ADV and LMV and FTC, or ADV and FTC and LMV and TFV. Referenceherein to “decreased” or “reduced” in relation to sensitivity to ADVand/or LMV and/or FTC and/or TFV includes and encompasses a complete orsubstantial resistance to the nucleoside or nucleotide analog or otheranti-HBV agents as well as partial resistance and includes a replicationrate or replication efficiency which is more than a wild-type in thepresence of a nucleoside or nucleotide analog or other anti-HBV agents.In one aspect, this is conveniently measured by an increase in viralload during treatment, or alternatively, there is no substantialdecrease in HBV DNA viral load from pre-treatment HBV DNA levels duringtreatment (i.e., non-response to treatment).

Before describing the present invention in detail, it is to beunderstood that unless otherwise indicated, the subject invention is notlimited to specific formulations of components, manufacturing methods,dosage regimens, or the like, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting.

It must be noted that, as used in the subject specification, thesingular forms “a”, “an” and “the” include plural aspects unless thecontext clearly dictates otherwise. Thus, for example, reference to “anucleoside or nucleotide analog” includes a single analog, as well astwo or more analogs; reference to “an HBV variant” includes reference totwo or more HBV variants; and so forth.

In describing and claiming the present invention, the followingterminology is used in accordance with the definitions set forth below.

The terms “analog”, “compound”, “active agent”, “pharmacologicallyactive agent”, “medicament”, “active” and “drug” are usedinterchangeably herein to refer to a chemical compound that induces adesired effect such as inhibit viral replication, infection,maintenance, assembly and/or the function of an enzyme such as HBV DNApolymerase. The terms also encompass pharmaceutically acceptable andpharmacologically active ingredients of those active agents specificallymentioned herein including but not limited to salts, esters, amides,prodrugs, active metabolites, analogs and the like. When the terms“analog”, “compound”, “active agent”, “pharmacologically active agent”,“medicament”, “active” and “drug” are used, then it is to be understoodthat this includes the active agent per se as well as pharmaceuticallyacceptable, pharmacologically active salts, esters, amides, prodrugs,metabolites, analogs, etc.

The present invention contemplates, therefore, compounds useful ininhibiting HBV replication, infection, maintenance, assembly and/or thefunction of an enzyme such as HBV DNA polymerase. Reference to an“analog”, “compound”, “active agent”, “pharmacologically active agent”,“medicament”, “active” and “drug” such as ADV, LMV, FTC and/or TFVincludes combinations of two or more actives such as ADV, LMV, TFV, orFTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV, FTC and TFV,FTC and LMV, or ADV and LMV and TFV, or ADV and FTC and TFV, TFV and FTCand LMV, ADV and LMV and FTC, or ADV and FTC and LMV and TFV. A“combination” also includes a two-part or more such as a multi-partanti-HBV therapeutic composition where the agents are providedseparately and given or dispensed separately or admixed together priorto dispensation.

The terms “effective amount” and “therapeutically effective amount” ofan agent as used herein mean a sufficient amount of the agent to providethe desired therapeutic or physiological effect of inhibiting HBVreplication, infection, maintenance, assembly and/or the function of anenzyme such as HBV DNA polymerase. Furthermore, an “effectiveHBV-inhibiting amount” or “effective symptom-ameloriating amount” of anagent is a sufficient amount of the agent to directly or indirectlyinhibit replication, infection, maintenance, assembly and/or thefunction of an enzyme such as HBV DNA polymerase. Undesirable effects,e.g. side effects, are sometimes manifested along with the desiredtherapeutic effect; hence, a practitioner balances the potentialbenefits against the potential risks in determining what is anappropriate “effective amount”. The exact amount required will vary fromsubject to subject, depending on the species, age and general conditionof the subject, mode of administration and the like. Thus, it may not bepossible to specify an exact “effective amount”. However, an appropriate“effective amount” in any individual case may be determined by one ofordinary skill in the art using only routine experimentation.

By “pharmaceutically acceptable” carrier, excipient or diluent is meanta pharmaceutical vehicle comprised of a material that is notbiologically or otherwise undesirable, i.e. the material may beadministered to a subject along with the selected active agent withoutcausing any or a substantial adverse reaction. Carriers may includeexcipients and other additives such as diluents, detergents, coloringagents, wetting or emulsifying agents, pH buffering agents,preservatives, and the like.

Similarly, a “pharmacologically acceptable” salt, ester, emide, prodrugor derivative of a compound as provided herein is a salt, ester, amide,prodrug or derivative that this not biologically or otherwiseundesirable.

The terms “treating” and “treatment” as used herein refer to reductionin severity and/or frequency of symptoms, elimination of symptoms and/orunderlying cause, prevention of the occurrence of symptoms and/or theirunderlying cause, and improvement or remediation of damage in relationto HBV infection. Thus, for example, “treating” a patient involvesprevention of HBV infection as well as treatment of a clinically HBVsymptomatic individual by inhibiting HBV replication, infection,maintenance, assembly and/or the function of an enzyme such as HBV DNApolymerase. Thus, for example, the present method of “treating” apatient with HBV infection or with a propensity for one to developencompasses both prevention of HBV infection as well as treating HBVinfection or symptoms thereof. In any event, the present inventioncontemplates the treatment or prophylaxis of HBV infection.

“Patient” as used herein refers to an animal, preferably a mammal andmore preferably a primate including a lower primate and even morepreferably, a human who can benefit from the formulations and methods ofthe present invention. A patient regardless of whether a human ornon-human animal may be referred to as an individual, subject, animal,host or recipient. The compounds and methods of the present inventionhave applications in human medicine, veterinary medicine as well as ingeneral, domestic or wild animal husbandry. For convenience, an “animal”includes an avian species such as a poultry bird (including ducks,chicken, turkeys and geese), an aviary bird or game bird. The conditionin a non-human animal may not be a naturally occurring HBV infection butHBV-like infection may be induced.

As indicated above, the preferred animals are humans, non-human primatessuch as marmosets, baboons, orangutans, lower primates such as tupia,livestock animals, laboratory test animals, companion animals or captivewild animals. A human is the most preferred target. However, non-humananimal models may be used.

Examples of laboratory test animals include mice, rats, rabbits, guineapigs and hamsters. Rabbits and rodent animals, such as rats and mice,provide a convenient test system or animal model as do primates andlower primates. Livestock animals include sheep, cows, pigs, goats,horses and donkeys. Non-mammalian animals such as avian species,zebrafish, amphibians (including cane toads) and Drosophila species suchas Drosophila melanogaster are also contemplated. Instead of a liveanimal model, a test system may also comprise a tissue culture system.

Accordingly, one aspect of the present invention is directed to anisolated HBV variant wherein said variant comprises a nucleotidemutation in a gene encoding a DNA polymerase resulting in at least oneamino acid addition, substitution and/or deletion to said DNA polymeraseand wherein said variant exhibits decreased sensitivity to ADV, LMV,TFV, or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV, FTCand TFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTC and TFV,TFV and FTC and LMV, or ADV and LMV and FTC, ADV and FTC and LMV and TFVand/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combination thereof.

HBV is a member of the Hepadnaviridae that includes also avian hepatitisviruses such as Duck hepatitis B virus (DHBV) and hepatitis viruses frommammals such as woodchuck hepatitis virus (WHV). These viruses havesimilarity to HBV and may be used in in vitro and in vivo or animalmodel systems to investigate the equivalent HBV mutants and anti-viralsensitivity to ADV, LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMVand TFV, FTC and ADV, FTC and TFV, FTC and LMV, or ADV and LMV and TFV,or ADV and FTC and TFV, TFV and FTC and LMV, ADV and LMV and FTC, or ADVand FTC and LMV and TFV,

An “anti-HBV agent” includes a nucleoside or nucleotide analog, protein,chemical compound, RNA or DNA or RNAi or siRNA oligonucleotide.

Preferably, the decreased sensitivity is in respect of ADV.Alternatively, the decreased sensitivity is in respect of LMV.Alternatively, the decreased sensitivity is in respect of TFV.Alternatively, the decreased sensitivity is in respect of FTC.Alternatively, the decreased sensitivity is in respect of ADV and LMV.Alternatively, the decreased sensitivity is in respect of ADV and TFV.Alternatively, the decreased sensitivity is in respect of LMV and TFV.Alternatively, the decreased sensitivity is in respect of ADV and FTC.Alternatively, the decreased sensitivity is in respect to FTC and TFV.Alternatively, the decreased sensitivity is in respect of FTC and LMV.Alternatively, the decreased sensitivity is in respect of ADV and LMVand TFV. Alternatively, the decreased sensitivity is in respect to ADVand TFV and FTC. Alternatively, the decreased sensitivity is in respectto LMV and TFV and FTC. Alternatively, the decrease sensitivity is inrespect of ADV and LMV and FTC. Alternatively, the decreased sensitivityis in respect of ADV and FTC and TFV and LMV.

Reference herein to “anti-HBV agents” includes nucleoside and nucleotideanalogs as well as immunological reagents (e.g. antibodies to HBVsurface components) and chemical, proteinaceous and nucleic acid agentswhich inhibit or otherwise interfere with viral replication,maintenance, infection, assembly or release. Reference herein to“nucleic acid” includes reference to a sense or antisense molecule, RNAor DNA, oligonucleotides and RNAi and siRNA molecules and complexescontaining same.

In addition to a mutation in the gene encoding DNA polymerase, due tothe overlapping nature of the HBV genome (FIG. 1), a correspondingmutation may also occur in the gene encoding the S gene encoding thesurface antigen (HBsAg) resulting in reduced interactivity ofimmunological reagents such as antibodies and immune cells to HBsAg. Thereduction in the interactivity of immunological reagents to a viralsurface component generally includes the absence of immunological memoryto recognize or substantially recognize the viral surface component. Thepresent invention extends, therefore, to an HBV variant exhibitingdecreased sensitivity to ADV, LMV, TFV, or FTC, or ADV and LMV, ADV andTFV, LMV and TFV, FTC and ADV, FTC and TFV, FTC and LMV, or ADV and LMVand TFV, or ADV and FTC and TFV, TFV and FTC and LMV, ADV and LMV andFTC, and/or ADV and FTC and LMV and TFV or a reduced interactivity of animmunological reagent to HBsAg wherein the variant is selected forfollowing ADV and/or LMV combination or sequential treatment. The term“sequential” in this respect means ADV followed by LMV and/or TFV,and/or FTC, LMV followed by ADV and/or TFV, and/or FTC, or multiplesequential administrations of each of ADV, LMV and/or TFV, and/or FTC.

A viral variant may, therefore, carry mutation only in the DNApolymerase gene or both in the DNA polymerase gene and the S gene. Theterm “mutation” is to be read in its broadest context and includesmultiple mutations.

The present invention extends to a Mutation and any domain of the HBVDNA polymerase and in particular regions F and G, and domains A throughto E provided said mutation leads to decreased sensitivity to ADV and/orLMV and/or TFV or combinations thereof. Regions F and G of the HBV DNApolymerase is defined by the amino acid sequence set forth in Formula Ibelow [SEQ ID NO:1]:

FORMULA I L, X₁, X₂, D, W, G, P, C, X₃, X₄, H, G, X₅, H, X₆, I, R, B₇,P, R, T, P, X₈, R, V, X₉, G, G, V, F, L, V, D, K, N, P, H, N, T, X₁₀, E,S, X₁₁, L, X₁₂, V, D, F, S, Q, F, S, R, G, X₁₃, X₁₄, X₁₅, V, P, K, F, A,V, P, N, L, X₁₆, S, L, T, N, L, L, S*wherein:X₁ is L, or R, or IX₂ is E, or DX₃ is T, or D, or A, or N, or YX₄ is E, or DX₅ is E, or K, or QX₆ is H, or R, or N,X₇ is I, or TX₈ is A, or SX₉ is T or RX₁₀ is A, or T, or SX₁₁ is R, or TX₁₂ is V, or GX₁₃ is S, or I, or T, or N, or VX₁₄ is T, or S, or H, or YX₁₅ is R, or H, or K, or QX₁₆ is Q, or P;and wherein S* is designated as amino acid 74.

In this specification, reference is particularly made to the conservedregions of the DNA polymerase as defined by domains A to E. Regions A toE are defined by the amino acid sequence set forth in Formula II below[SEQ ID NO:2] (and in Australian Patent No. 734831):

FORMULA II S X₁ L S W L S L D V S A A F Y H X₂ P L H P A A M P H L L X₃G S S G L X₄ R Y V A R L S S X₅ S X₆ X₇ X N X₈ Q X₉ X₁₀ X X X X₁₁ L HX₁₂ X₁₃ C S R X₁₄ L Y V S L X₁₅ L L Y X₁₆ T X₁₇ G X₁₈ K L H L X₁₉ X₂₀ HP I X₂₁ L G F R K X₂₂ P M G X₂₃ G L S P F L L A Q F T S A I X₂₄ X₂₅ X₂₆X₂₇ X₂₈ R A F X₂₉ H C X₃₀ X₃₁ F X₃₂ Y M* D D X₃₃ V L G A X₃₄ X₃₅ X₃₆ X₃₇H X₃₈ E X₃₉ L X₄₀ X₄₁ X₄₂ X₄₃ X₄₄ X₄₅ X₄₆ L L X₄₇ X₄₈ G I H L N P X₄₉ KT K R W G Y S L N F M G Y X₅₀ I Gwherein:X is any amino acidX₁ is N or D;X₂ is 1 or 1′;X₃ is I or V;X₄ is S or D;X₅ is T or N;X₆ is R or N;X₇ is N or I;X₈ is N or Y or H;X₉ is H or Y;X₁₀ is G or R;X₁₁ is D or N;X₁₂ is D or N;X₁₃ is S or Y;X₁₄ is N or Q;X₁₅ is L or M;X₁₆ is K or Q;X₁₇ is Y or F;X₁₈ is R or W;X₁₉ is Y or L;X₂₀ is or A;X₂₁ is I or V;X₂₂ is I or L;X₂₃ is V or G;X₂₄ is C or L;X₂₅ is A or S;X₂₆ is V or M;X₂₇ is V or T;X₂₈ is R or C;X₂₉ is F or P;X₃₀ is L or V;X₃₁ is A or V;X₃₂ is S or A;X₃₃ is V or L or M;X₃₄ is K or R;X₃₅ is S or T;X₃₆ is V or G;X₃₇ is Q or E;X₃₈ is L or S or R;X₃₉ is S or F;X₄₀ is F or Y;X₄₁ is T or A;X₄₂ is A or S;X₄₃ is V or I;X₄₄ is T or C;X₄₅ is N or S;X₄₆ is F or V;X₄₇ is S or D;X₄₈ is L or V;X₄₉ is N or Q;X₅₀ is V or I; andM* is amino acid 204;and wherein the first S is designated as amino acid 75.

Preferably, the mutation results in an altered amino acid sequence inany one or more of domains F and G, and domains A through to E orregions proximal thereto of the HBV DNA polymerase.

Another aspect of the present invention provides an HBV variantcomprising a mutation in an overlapping open reading frame in its genomewherein said mutation is in a region defined by one or more of domains Fand G, and domains A through to E of HBV DNA polymerase and wherein saidvariant exhibits decreased sensitivity to ADV, LMV, TFV, or FTC, or ADVand LMV, ADV and TFV, LMV and TFV, FTC and ADV, FTC and TFV, FTC andLMV, or ADV and LMV and TFV, or ADV and FTC and TFV, TFV and FTC andLMV, ADV and LMV and FTC, or ADV and FTC and LMV and TFV and/oroptionally other nucleoside or nucleotide analogs or other anti-HBVagents.

In a related embodiment, there is provided an HBV variant comprising amutation in the nucleotide sequence encoding a DNA polymerase resultingin an amino acid addition, substitution and/or deletion in said DNApolymerase in one or more amino acids as set forth in Formula I [SEQ IDNO:1] and/or Formula II [SEQ ID NO:2]:

FORMULA I L, X₁, X₂, D, W, G, P, C, X₃, X₄, H, G, X₅, H, X₆, I, R, X₇,P, R, T, P, X₈, R, V, X₉, G, G, V, F, L, V, D, K, N, P, H, N, T, X₁₀, E,S, X₁₁, L, X₁₂, V, D, F, S, Q, F, S, R, G, X₁₃, X₁₄, X₁₅, V, S, W, P, K,F, A, V, P, N, L, X₁₆, S, L, T, N, L, L, S*wherein:X₁ is L, or R, or IX₂ is E, or DX₃ is T, or D, or A, or N, or YX₄ is E, or DX₅ is E, or K, or QX₆ is H, or R, or N,X₇ is I, or TX₈, is A, or SX₉ is T or RX₁₀ is A, or T, or SX₁₁ is R, or TX₁₂ is V, or GX₁₃ is S, or I, or T, or N, or VX₁₄ is T, or S, or H, or YX₁₅ is R, or H, or K, or QX₁₆ is Q, or P;and

FORMULA II S X₁ L S W L S L D V S A A F Y H X₂ P L H P A A M P H L L X₃G S S G L X₄ R Y V A R L S S X₅ X₆ X₇ X N X₈ Q X₉ X₁₀ X X X X₁₁ L H X₁₂X₁₃ C S R X₁₄ L Y V S L X₁₅ L L Y X₁₆ T X₁₇ G X₁₈ K L H L X₁₉ X₂₀ H P IX₂₁ L G F R K X₂₂ P M G X₂₃ G L S P F L L A Q F T S A I X₂₄ X₂₅ X₂₆ X₂₇X₂₈ R A F X₂₉ H C X₃₀ X₃₁ F X₃₂ Y M* D D X₃₃ V L G A X₃₄ X₃₅ X₃₆ X₃₇ HX₃₈ X₃₉ L X₄₀ X₄₁ X₄₂ X₄₃ X₄₄ X₄₅ X₄₆ L L X₄₇ X₄₈ G I H L N P X₄₉ K T KR W G Y S L N F M G Y X₅₀ I Gwherein:X is any amino acidX₁ is N or D;X₂ is I or P;X₃ is or V;X₄ is S or D;X₅ is T or N;X₆ is R or N;X₇ is N or I;X₈ is N or Y or H;X₉ is H or Y;X₁₀ is G or R;X₁₁ is D or N;X₁₂ is D or N;X₁₃ is S or Y;X₁₄ is N or Q;X₁₅ is L or M;X₁₆ is K or Q;X₁₇ is Y or F;X₁₆ is R or W;X₁₉ is Y or L;X₂₀ is S or A;X₂₁ is I or V;X₂₂ is I or L;X₂₃ is V or G;X₂₄ is C or L;X₂₅ is A or S;X₂₆ is V or M;X₂₇ is V or T;X₂₈ is R or C;X₂₉ is F or P;X₃₀ is L or V;X₃₁ is A or V;X₃₂ is S or A;X₃₃ is V or L or M;X₃₄ is K or R;X₃₅ is S or T;X₃₆ is V or G;X₃₇ is Q or E;X₃₈ is L or S or R;X₃₉ is S or F;X₄₀ is F or Y;X₄₁ is T or A;X₄₂ is A or S;X₄₃ is V or I;X₄₄ is T or C;X₄₅ is N or S;X₄₆ is F or V;X₄₇ is S or D;X₄₈ is L or V;X₄₉ is N or Q;X₅₀ is V or I; andM* is amino acid 204;and wherein S* in Formula I is designated as amino acid 74 and the firstS in Formula II is designated as amino acid 75;and wherein said variant exhibits decreased sensitivity to ADV, LMV,TFV, or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV, FTCand TFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTC and TFV,TFV and FTC and LMV, ADV and LMV and FTC, or ADV and FTC and LMV and TFVand/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combination thereof. Preferably, the decreasedsensitivity is to ADV or to both ADV and LMV or to one or both of ADVand/or LMV and/or TFV and for FTC.

Another preferred aspect of the present invention contemplates an HBVvariant comprising a mutation in the nucleotide sequence encoding HBsAgresulting in an amino acid addition, substitution and/or deletion insaid HBsAg in a region corresponding to the amino acid sequence setforth in Formulae I and II wherein said variant exhibits decreasedsensitivity to ADV, LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMVand TFV, FTC and ADV, FTC and TFV, FTC and LMV, or ADV and LMV and TFV,or ADV and FTC and TFV, TFV and FTC and LMV, ADV and LMV and FTC, or ADVand FTC and LMV and TFV and/or optionally other nucleoside or nucleotideanalogs or other anti-HBV agents or combination thereof.

More particularly, the present invention provides a variant HBVcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion or atruncation compared to a surface antigen from a reference or wild-typeHBV and wherein an antibody generated to the reference or wild-typesurface antigen exhibits reduced capacity for neutralizing said HBVvariant, said variant selected by exposure of a subject to ADV, LMV,TFV, or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV, FTCand TFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTC and TFV,TFV and FTC and LMV, ADV and LMV and FTC, or ADV and FTC and LMV and TFVand/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combination thereof.

The term “combination therapy” means that both combinations of ADV, LMV,FTC and/or TFV are co-administered in the same composition orsimultaneously in separate compositions. The term “sequential therapy”means that the two agents are administered within seconds, minutes,hours, days or weeks of each other and in either order. Sequentialtherapy also encompasses completing a therapeutic course with one orother of ADV, LMV, FTC or TFV and then completing a second or third orsubsequent therapeutic courses with the other of ADV, LMV, FTC or TFV.

Accordingly, another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV therapy or therapy by one or moreother nucleoside or nucleotide analogs or other anti-HBV agents.

Another aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to LMV therapy or therapy by one or moreother nucleoside or nucleotide analogs or other anti-HBV agents.

Yet another aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to FTC therapy or therapy by one or moreother nucleoside or nucleotide analogs or other anti-HBV agents.

Still another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to TFV therapy or therapy by one or moreother nucleoside or nucleotide analogs or other anti-HBV agents.

Even yet another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV and LMV therapy or therapy by one ormore other nucleoside or nucleotide analogs or other anti-HBV agents.

Even still another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV and TFV therapy or therapy by one ormore other nucleoside or nucleotide analogs or other anti-HBV agents.

A further aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to LMV and TFV therapy or therapy by one ormore other nucleoside or nucleotide analogs or other anti-HBV agents.

Another aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV and FTC therapy or therapy by one ormore other nucleoside or nucleotide analogs or other anti-HBV agents.

Yet another aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to TFV and FTC therapy or therapy by one ormore other nucleoside or nucleotide analogs or other anti-HBV agents.

Still another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to FTC and LMV therapy or therapy by one ormore other nucleoside or nucleotide analogs or other anti-HBV agents.

Even yet another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV, LMV and TFV therapy or therapy byone or more other nucleoside or nucleotide analogs or other anti-HBVagents.

Even still another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV, LMV and TFV therapy or therapy byone or more other nucleoside or nucleotide analogs or other anti-HBVagents.

A further aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV, LMV and FTC therapy or therapy byone or more other nucleoside or nucleotide analogs or other anti-HBVagents.

Another aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to FTC, LMV and TFV therapy or therapy byone or more other nucleoside or nucleotide analogs or other anti-HBVagents.

Yet another aspect of the present invention contemplates an HBV variantcomprising a surface antigen having an amino acid sequence with a singleor multiple amino acid substitution, addition and/or deletion ortruncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV, FTC and TFV therapy or therapy byone or more other nucleoside or nucleotide analogs or other anti-HBVagents.

Still yet another aspect of the present invention contemplates an HBVvariant comprising a surface antigen having an amino acid sequence witha single or multiple amino acid substitution, addition and/or deletionor truncation compared to the pretreatment HBV and wherein the surfaceantigen of the variant HBV exhibits an altered immunological profilecompared to the pretreatment HBV where the said variant HBV is selectedfor by exposure of a subject to ADV, LMV, FTC and TFV therapy or therapyby one or more other nucleoside or nucleotide analogs or other anti-HBVagents.

Preferably, the variants are in isolated form such that they haveundergone at least one purification step away from naturally occurringbody fluid. Alternatively, the variants may be maintained in isolatedbody fluid or may be in DNA form. The present invention alsocontemplates infectious molecular clones comprising the genome or partsthereof from a variant HBV. Furthermore, the present invention providesisolated components from the variant HBVs such as but not limited to anisolated HBsAg. Accordingly, the present invention provides an isolatedHBsAg or a recombinant form thereof or derivative or chemical equivalentthereof, said HBsAg being from a variant HBV selected by exposure of asubject to one or more of ADV, LMV, FTC and/or TFV or optionally one ormore nucleoside or nucleotide analogs or other anti-HBV agents.

More particularly, yet another aspect of the present invention isdirected to an isolated variant HBsAg or a recombinant or derivativeform thereof or a chemical equivalent thereof wherein said HBsAg or itsrecombinant or derivative form or its chemical equivalent exhibits analtered immunological profile compared to an HBsAg from a reference HBV,said HBsAg being from a variant HBV selected by exposure of a subject toone or more of ADV, LMV, FTC and/or TFV or optionally one or morenucleoside or nucleotide analogs or other anti-HBV agents.

Even more particularly, the present invention provides an isolatedvariant HBsAg or a recombinant or derivative form thereof or a chemicalequivalent thereof wherein said HBsAg or its recombinant or derivativeform or its chemical equivalent comprises an amino acid sequence with asingle or multiple amino acid substitution, addition and/or deletion ora truncation compared to an HBsAg from a reference HBV and wherein aneutralizing antibody directed to a reference HBV exhibits no or reducedneutralising activity to an HBV carrying said variant HBsAg, said HBsAgbeing from a variant HBV selected by exposure of a subject to one ormore of ADV, LMV, FTC and/or TFV or optionally one or more nucleoside ornucleotide analogs or other anti-HBV agents.

Preferred mutations in the HBV DNA polymerase include variants selectedfrom patients with HBV recurrence following ADV, LMV, TFV, or FTC, orADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV, FTC and TFV, FTC andLMV, or ADV and LMV and TFV, or ADV and FTC and TFV, TFV and FTC andLMV, ADV and LMV and FTC, or ADV and FTC and LMV and TFV treatment.Nucleoside or nucleotide analog or other anti-HBV agents treatment mayoccur in relation to a transplantation procedure (e.g. bone marrowtransplantation (BMT) or OLT) or following treatment of patientsdiagnosed with hepatitis. Following selection of variants, viral loadsare obtainable at levels similar to pre-treatment levels or areincreasing while on therapy.

Preferred mutations include, in one embodiment, rtS21A, rtL122F,rtN124H, rtH126R, rtT28N, rtP130Q, rtD131N and rtY135C; in anotherembodiment, rt/N/S/T/I/V53D, rtY126Q, rtL180M, rtS202G, rtI204V andrtI235I/M; in a further embodiment, rtN53D, rtY54H, rtS57P, rtL91I,rtS116P, rtF122L, rtY124H, rtV134D, rtY141Y/F, rtL145M, rtF151F/Y,rtA181T, rtK212R, rtL217R, rtS219A, rtN236T and rtN238D; in yet anotherembodiment, rtS78T, rtV84M, rtY126C, rtV191I, rtM204I and rtV214A; instill another embodiment, rtH90D, and rtL/F108L; in even yet anotherembodiment, rtL157L/M, rtA181V and rtV207I; in still yet anotherembodiment, rtL80V, rtP109S, rtI163V, rtL229M and rtN/H/A/S/Q238K; inanother embodiment, rtS78S/T, rtN118N/S, rtN139N/K, rtV142E, rtA181A/T,rtI204M, rtQ/F/S/Stop215Q, rtE218K/E and rtN238N/H; in a furtherembodiment, rtK32, rtN33, rtP34, rtH35 and rtT37; in yet anotherembodiment, rtP59, rtK60, rtF61, rtA62 and rtV63; in still anotherembodiment, rtD83, rtV84, rtS85, rtA86, rtY89, rtH90 and rtI/L91; ineven yet another embodiment, rtP177, rtF178, rtL179, rtL180, rtA181,rtQ182, rtF183 and rtT184; in still yet another embodiment, rtM204 andrtY203; in another embodiment, rt235, rt236, rt237, rt238 and rt239; ina further embodiment, rt247, rt248, rt249, rt250 and rt251; in yetanother embodiment.

K32M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;N33D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/deletion;P34S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;H35I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;T37W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/deletion;P59S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;K60M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;F61P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/deletion;A62R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/deletion;V63A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;D83C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/deletion;V84A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;S85T/W/Y/V/N/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/ deletion;A86R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/YV/deletion;Y89V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/deletion;H90I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;I/L91K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/deletion;P177S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;F178P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;L179K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;L180K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;A181R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/YV/deletion;Q183E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;F183P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;T184W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;Y203V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/ deletion;M204F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;L235K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N236D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;T237W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;P237S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;N238D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H238I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;A238R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;S239T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/ deletion;Q238E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;K239M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/ deletion;L247K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N248D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H248I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;F249P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;M250F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;G251H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E; andV251A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y.

Reference above to “deletion” means that the first mentioned amino acidbefore the residue number has been deleted.

Such HBV variants are proposed to exhibit a decreased sensitivity toADV, LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC andADV, FTC and TFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTCand TFV, TFV and FTC and LMV, ADV and LMV and FTC, or ADV and FTC andLMV and TFV and/or optionally other nucleoside or nucleotide analogs orother anti-HBV agents or combination thereof. It should be noted thatthe nomenclature system for amino acid positions is based on themethionine residues in the YMDD motif being designated codon rtM204.This numbering system is different to that iii Australian Patent No.734831 where the methionine residue in the YMDD motif within thepolymerase gene is designated codon 550. In this regard, rtL180M andrtM204V correspond to L526M and M550V, respectively, in AustralianPatent No. 734831. Corresponding mutations may also occur in envelopegenes such as in one or more of PreS1, PreS2 and S. The mutations in Sgene encoding HBsAg at sT118R, sP120T, sS143S/T, sD144A or sI195M alsoresult in mutation in the in the polymerase gene rtY126C, rtT128N,rtF151S/F or rtM204V respectively.

Another potential mode of action of ADV and other acyclic nucleosidephosphonates is that of immune stimulation (Calio et al., Antiviral Res.23: 77-89, 1994). A number of mutations resulted in changes in theenvelope gene detected in HBV variants which may be associated withimmune escape. These changes include sT118R, sP120T, sS126T, sM133T,sM133L/M, sF134V, sS143S/T, sD144A, sG145A and/or sW172STOP.

HBV encoding the mutation at codon sG145R is a well characterizedvaccine escape mutant, although the envelope protein from HBV encodingthe mutation at sG145A does not have the same antigen/antibody bindingcharacteristics as the sG145R. This mutation was detected in HBVisolated from patient C in conjunction with mutations at codons 143 and144.

The identification of the variants of the present invention permits thegeneration of a range of assays to detect such variants. The detectionof such variants may be important in identifying resistant variants todetermine the appropriate form of chemotherapy and/or to monitorvaccination protocols, or develop new or modified vaccine preparations.

Still another aspect of the present invention contemplates a method fordetermining the potential for an HBV to exhibit reduced sensitivity toADV, LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC andADV, FTC and TFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTCand TFV, TFV and FTC and LMV, ADV and LMV and FTC, or ADV and FTC andLMV and TFV and/or optionally other nucleoside or nucleotide analogs orother anti-HBV agents, said method comprising isolating DNA orcorresponding mRNA from said HBV and screening for a mutation in thenucleotide sequence encoding HBV DNA polymerase resulting in at leastone amino acid substitution, deletion and/or addition in any one or moreof domains F and G, and A domains through to E or a region proximalthereto of said DNA polymerase and associated with resistance ordecreased sensitivity to ADV, LMV, TFV, or FTC, or ADV and LMV, ADV andTFV, LMV and TFV, FTC and ADV, FTC and TFV, FTC and LMV, or ADV and LMVand TFV, or ADV and FTC and TFV, TFV and FTC and LMV, ADV and LMV andFTC, or ADV and FTC and LMV and TFV and/or optionally other nucleosideor nucleotide analogs or other anti-HBV agents wherein the presence ofsuch a mutation is an indication of the likelihood of resistance to saidADV, LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC andADV, FTC and TFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTCand TFV, TFV and FTC and LMV, ADV and LMV and FTC, or ADV and FTC andLMV and TFV and/or optionally other nucleoside or nucleotide analogs orother anti-HBV agents.

Preferably, the assay detects one or more of the following mutations: inone embodiment, rtS21A, rtL122F, rtN124H, rtH126R, rtT28N, rtP130Q,rtD131N and rtY135C; in another embodiment, rt/N/S/T/I/V53D, rtY126Q,rtL180M, rtS202G, rtI204V and rtI235I/M; in a further embodiment,rtN53D, rtY54H, rtS57P, rtL91I, rtS116P, rtF122L, rtY124H, rtV134D,rtY141Y/F, rtL145M, rtF151F/Y, rtA181T, rtK212R, rtL217R, rtS219A,rtN236T and rtN238D; in yet another embodiment, rtS78T, rtV84M, rtY126C,rtV191I, rtM204I and rtV214A; in still another embodiment, rtH90D andrtL/F108L; in even yet another embodiment, sP120T, sM125T and sT127A; instill yet another embodiment, sT118R, sM133T, SF134V, sI195M, sS207R andsY225Y/C; in another embodiment, sS126T, sM133L/M, sS143S/T, sD144A,sG145A and sW172Stop; in a further embodiment, sN40S, sC69STOP, sM75I,sL88P, sT118A, sW182Stop, sW196L, sY206H and sY225F; in yet anotherembodiment, s181M and sP214Q; in still another embodiment, sF83S, sL173Fand sW199L; in yet another embodiment, sI126T, sK160R, sS174N, sA184V,sW196L, sS210N, sF/C220L and sY221C; in still another embodiment,sC69Stop/C, sC76Y, sI110V/I, sY134N, sW172Stop/W, sW196Stop, sS207R; ineven still another embodiment, rtK32, rtN33, rtP34, rtH35 and rtT37); inanother embodiment, rtP59, rtK60, rtF61, rtA62 and rtV63); in a furtherembodiment, rtD83, rtV84, rtS85, rtA86, rtY89, rtH90 and rtI/L91); inyet another embodiment, rtP177, rtF178, rtL179, rtL180, rtA181, rtQ182,rtF183 and rtT184; in still another embodiment, rtM204 and rtY203; ineven yet another embodiment, rt235, rt236, rt237, rt238 and rt239 and ineven still another embodiment, rt247, rt248, rt249, rt250 and rt251 andin another embodiment,

K32M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;N33D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/deletion;P34S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M//deletionF;H35I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;T37W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/deletion;P59S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;K60M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;F61P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/deletion;A62R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/deletion;V63A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;D83C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/deletion;V84A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;S85T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/deletion;A86R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/YV/deletion;Y89V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/deletion;H90I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;I/L91K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/deletion;P177S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;F178P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;L179K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;L180K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;A181R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;Q183E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;F183P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;T184W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;Y203V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/ deletion;M204F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;L235K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N236D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;T237W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;P237S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;N238D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H23SI/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;A238R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/YV/ deletion;S239T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/ deletion;Q238E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;K239M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/ deletion;L247K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N248D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H248I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;F249P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;M250F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;G251H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/ deletion; andV251A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/ deletionor combinations thereof or an equivalent one or more other mutation isindicative of a variant wherein said variant exhibits a decreasedsensitivity to ADV, LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMVand TFV, FTC and ADV, FTC and TFV, FTC and LMV, or ADV and LMV and TFV,or ADV and FTC and TFV, TFV and FTC and LMV, ADV and LMV and FTC, or ADVand FTC and LMV and TFV and/or optionally other nucleoside or nucleotideanalogs or other anti-HBV agents or combination thereof.

Accordingly, another aspect of the present invention produces a methodfor determining whether an HBV strain exhibits reduced sensitivity to anucleoside or nucleotide analog or other anti-HBV agents, said methodcomprising isolating DNA or corresponding mRNA from said HBV andscreening for a mutation in the nucleotide sequence encoding the DNApolymerase and/or a corresponding region of the S gene, wherein thepresence of a mutation selected from, in one embodiment, rtS21A,rtL122F, rtN124H, rtH126R, rtT28N, rtP130Q, rtD131N and rtY135C; inanother embodiment, rt/N/S/T/I/V53D, rtY126Q, rtL180M, rtS202G, rtI204Vand rtI235I/M; in a further embodiment, rtN53D, rtY54H, rtS57P, rtL91I,rtS116P, rtF122L, rtY124H, rtV134D, rtY141Y/F, rtL145M, rtF151F/Y,rtA181T, rt212R, rtL217R, rtS219A, rtN236T and rtN238D; in yet anotherembodiment, rtS78T, rtV84M, rtY126C, rtV191I, rtM204I and rtV214A; instill another embodiment, rtH90D and rtL/F108L; in even yet anotherembodiment, sP120T, sM125T and sT127A; in still yet another embodiment,sT118R, sM133T, SF134V, sI195M, sS207R and sY225Y/C; in anotherembodiment, sS126T, sM133L/M, sS143S/T, sD144A, sG145A and sW172Stop; ina further embodiment, sN40S, sC69STOP, sM75I, sL88P, sT118A, sW182Stop,sW196L, sY206H and sY225F; in yet another embodiment, s181M and sP214Q;in still another embodiment, sF83S, sL173F and sW199L; in yet anotherembodiment, sI126T, sK160R, sS174N, sA184V, sW196L, sS210N, sF/C220L andsY221C; in still another embodiment, sC69Stop/C, sC76Y, sI110V/I,sY134N, sW172Stop/W, sW196Stop, sS207R; in even still anotherembodiment, rtK32, rtN33, rtP34, rtH35 and rtT37); in anotherembodiment, rtP59, rtK60, rtF61, rtA62 and rtV63); in a furtherembodiment, rtD83, rtV84, rtS85, rtA86, rtY89, rtH90 and rtI/L91); inyet another embodiment, rtP177, rtF178, rtL179, rtL180, rtA181, rtQ182,rtF183 and rtT184; in still another embodiment, rtM204 and rtY203; ineven yet another embodiment, rt235, rt236, rt237, rt238 and rt239 and ineven still another embodiment, rt247, rt248, rt249, rt250 and rt251; andin another embodiment,

K32M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;N33D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/deletion;P34S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;H35I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;T37W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/deletion;P59S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;K60M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;F61P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/deletion;A62R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/deletion;V63A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;D83C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/deletion;V84A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;S85T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/deletion;A86R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/deletion;Y89V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/deletion;H90I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;I/L91K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/deletion;P177S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;F178P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;L179K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;L180K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;A181R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;Q183E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;F183P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;T184W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;Y203V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/ deletion;M204F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;L235K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N236D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;T237W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;P237S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;N238D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H238I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;A238R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;S239T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/ deletion;Q238E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;K239M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/ deletion;L247K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N248D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H248I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;F249P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;M250F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;G251H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/ deletion; andV251A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/ deletionor combinations thereof or an equivalent one or more other mutation isindicative of a variant which exhibits a decreased sensitivity to ADV,LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV,FTC and TFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTC andTFV, TFV and FTC and LMV, ADV and LMV and FTC, or ADV and FTC and LMVand TFV and/or optionally other nucleoside or nucleotide analogs orother anti-HBV agents or combination thereof.

A further aspect of the present invention produces a method fordetermining whether an HBV strain exhibits reduced sensitivity to anucleoside or nucleotide analog or other anti-HBV agents, said methodcomprising isolating DNA or corresponding mRNA from said HBV andscreening for a mutation in the nucleotide sequence encoding the DNApolymerase and/or a corresponding region of the S gene, wherein thepresence of a mutation selected from, in one embodiment, rtS21A,rtL122F, rtN124H, rtH126R, rtT28N, rtP130Q, rtD131N and rtY135C; inanother embodiment, rt/N/S/T/I/V53D, rtY126Q, rtL180M, rtS202G, rtI204Vand rtI235I/M; in a further embodiment, rtN53D, rtY54H, rtS57P, rtL91I,rtS116P, rtF122L, rtY124H, rtV134D, rtY141Y/F, rtL145M, rtF151F/Y,rtA181T, rtK212R, rtL217R, rtS219A, rtN236T and rtN238D; in yet anotherembodiment, rtS78T, rtV84M, rtY126C, rtV191I, rtM204I and rtV214A; instill another embodiment, rtH90D and rtL/F108L; in even yet anotherembodiment, sP120T, sM125T and sT127A; in still yet another embodiment,sT118R, sM133T, SF134V, sI195M, sS207R and sY225Y/C; in anotherembodiment, sS126T, sM133L/M, sS143S/T, sD144A, sG145A and sW172Stop; ina further embodiment, sN40S, sC69STOP, sM75I, sL88P, sT118A, sW182Stop,sW196L, sY206H and sY225F; in yet another embodiment, s181M and sP214Q;in still another embodiment, sF83S, sL173F and sW199L; in yet anotherembodiment, sI126T, sK160R, sS174N, sA184V, sW196L, sS210N, sF/C220L andsY221C; in still another embodiment, sC69Stop/C, sC76Y, sI110V/I,sY134N, sW172Stop/W, sW196Stop, sS207R; in even still anotherembodiment, rtK32, rtN33, rtP34, rtH35 and rtT37); in anotherembodiment, rtP59, rtK60, rtF61, rtA62 and rtV63); in a furtherembodiment, rtD83, rtV84, rtS85, rtA86, rtY89, rtH90 and rtI/L91); inyet another embodiment, rtP177, rtF178, rtL179, rtL180, rtA181, rtQ182,rtF183 and rtT184; in still another embodiment, rtM204 and rtY203; ineven yet another embodiment, rt235, rt236, rt237, rt238 and rt239 and ineven still another embodiment, rt247, rt248, rt249, rt250 and rt251; andin another embodiment,

K32M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;N33D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/deletion;P34S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;H35I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;T37W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/deletion;P59S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;K60M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;F61P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/deletion;A62R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/deletion;V63A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;D83C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/deletion;V84A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;S85T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/deletion;A86R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/deletion;Y89V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/deletion;H90I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;I/L91K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/deletion;P177S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;F178P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;L179K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;L180K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;A181R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;Q183E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;F183P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;T184W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;Y203V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/ deletion;M204F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;L235K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N236D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;T237W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;P237S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;N238D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H238I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;A238R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;S239T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/ deletion;Q238E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;K239M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/ deletion;L247K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N248D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H248I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;F249P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;M250F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;G251H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/ deletion; andV251A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/ delectionor combinations thereof or an equivalent one or more other mutation isindicative of a variant which exhibits a decreased sensitivity to ADV,LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV,FTC and TFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTC andTFV, TFV and FTC and LMV, ADV and LMV and FTC, or ADV and FTC and LMVand TFV and/or optionally other nucleoside or nucleotide analogs orother anti-HBV agents or combination thereof.

The detection of HBV or its components in cells, cell lysates, culturedsupernatant fluid and bodily fluid may be by any convenient meansincluding any nucleic acid-based detection means, for example, bynucleic acid hybridization techniques or via one or more polymerasechain reactions (PCRs). The term “bodily fluid” includes any fluidderived from the blood, lymph, tissue or organ systems including serum,whole blood, biopsy and biopsy fluid, organ explants and organsuspension such as liver suspensions. The invention further encompassesthe use of different assay formats of said nucleic acid-based detectionmeans, including restriction fragment length polymorphism (RFLP),amplified fragment length polymorphism (AFLP), single-strand chainpolymorphism (SSCP), amplification and mismatch detection (AMD),interspersed repetitive sequence polymerase chain reaction (IRS-PCR),inverse polymerase chain reaction (iPCR) and reverse transcriptionpolymerase chain reaction (RT-PCR), amongst others. Other forms ofdetection include Northern blots, Southern blots, PCR sequencing,antibody procedures such as ELISA, Western blot andimmunohistochemistry. A particularly useful assay includes the reagentsand components required for immobilized oligonucleotide- oroligopeptide-mediated detection systems.

One particularly useful nucleic acid detection system is the reversehybridization technique. In this technique, DNA from an HBV sample isamplified using a biotin or other ligand-labeled primer to generate alabeled amplificon. Oligonucleotides immobilized to a solid support suchas a nitrocellulose film are then used to capture amplified DNA byhybridization. Specific nucleic acid fragments are identified via biotinor the ligand. Generally, the labeled primer is specific for aparticular nucleotide variation to be detected. Amplification occursonly if the variation to be detected is present. There are many forms ofthe reverse hybridization assay and all are encompassed by the presentinvention.

Detecting HBV replication in cell culture is particularly useful.

This and other aspects of the present invention is particularly amenableto microarray analysis such as to identify oligonucleotides includingsense and antisense molecules, RNAi or siRNA molecules or DNA orRNA-binding molecules which down-regulate genomic sequences ortranscripts of HBV. Microarray analysis may also be used to identifyparticular mutations in the HBV genome such as within the HBV DNApolymerase-coding region or the HBsAg-coding region.

Another aspect of the present invention contemplates a method fordetecting an agent which exhibits inhibitory activity to an HBV by:

-   -   generating a genetic construct comprising a replication        competent-effective amount of the genome from the HBV contained        in a plasmid vector and then transfecting said cells with said        construct;    -   contacting the cells, before, during and/or after transfection,        with the agent to be tested;    -   culturing the cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to said agents; and    -   then subjecting the cells, cell lysates or culture supernatant        fluid to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of the agent.

In a preferred embodiment, the plasmid vector may include genes encodingpart or all of other viral vectors such as baculovirus or adenovirus(Ren and Nassal, 2001, supra) and the method comprises:

-   -   generating a genetic construct comprising a replication        competent-effective amount of the genome from the HBV contained        in or fused to an amount of a baculovirus genome or adenovirus        genome effective to infect cells and then infecting said cells        with said construct;    -   contacting the cells, before, during and/or after infection,        with the agent to be tested;    -   culturing the cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to said agent; and    -   then subjecting the cells, cell lysates or culture supernatant        fluid to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of the agent.

In an alternative embodiment, the method comprises:

-   -   generating a continuous cell line comprising an infectious copy        of the genome of the HBV in a replication competent effective        amount such that said infectious HBV genome is stably integrated        into said continuous cell line such as but not limited to 2.2.15        or AD;    -   contacting the cells with the agent to be tested;    -   culturing the cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to the agent; and    -   then subjecting the cells, cell lysates or culture supernatant        fluid to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of the agent.

The above-mentioned methods are particularly useful in identifying ordeveloping agents against HBV variants such as those carrying mutations,in one embodiment, rtS21A, rtL122F, rtN124H, rtH126R, rtT28N, rtP130Q,rtD131N and rtY135C; in another embodiment, rt/N/S/T/I/V53D, rtY126Q,rtL180M, rtS202G, rtI204V and rtI235UM; in a further embodiment, rtN53D,rtY54H, rtS57P, rtL91I, rtS116P, rtF122L, rtY124H, rtV134D, rtY141Y/F,rtL145M, rtF151F/Y, rtA181T, rtK212R, rtL217R, rtS219A, rtN236T andrtN238D; in yet another embodiment, rtS78T, rtV84M, rtY126C, rtV191I,rtM204I and rtV214A; in still another embodiment rtH90D and rtL/F108L;in even yet another embodiment, rtL157L/M, rtA181V and rtV207I; in evenstill another embodiment, rtL80V, rtP109S, rtI163V, rtL229M andrtN/H/A/S/Q238K; in another embodiment, rtS78S/T, rtN118N/S, rtN139N/K,rtV142E, rtA181A/T, rtI204M, rtQ/P/S/Stop215Q, rtE218K/E and rtN238N/H;in a further embodiment, sP120T, sM125T and sT127A; in yet anotherembodiment, sT118R, sM133T, SF134V, sI195M, sS207R and sY225Y/C; instill another embodiment, sS126T, sM133L/M, sS143S/T, sD144A, sG145A andsW172Stop; in even yet another embodiment, sN40S, sC69Stop, sM75I,sL88P, sT118A, sW182STOP, sW196L, sY206H and sY225F; in even stillanother embodiment, s181M and sP214Q; in another embodiment, sF83S,sL173F and sW199L; in a further embodiment, sI126T, sK160R, sS174N,sA184V, sW196L, sS210N, sF/C220L and sY221C; in yet another embodiment,sC69Stop/C, sC76Y sI110V/I, sY134N, sW172Stop/W, sW196Stop and sS207R;in still another embodiment, rtK32, rtN33, rtP34, rtH35 and rtT37; ineven yet another embodiment, rtP59, rtK60, rtF61, rtA62 and rtV63; ineven still another embodiment, rtD83, rtV84, rtS85, rtA86, rtY89, rtH90and rtI/L91; in another embodiment, rtP177, rtF178, rtL179, rtL180,rtA181, rtQ182, rtF183 and rtT184; in a further embodiment, rtM204 andrtY203; in yet another embodiment, rt235, rt236, rt237, rt238 and rt239in still another embodiment, rt247, rt248, rt249, rt250 and rt251; andin even yet another embodiment,

K32M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;N33D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/deletion;P34S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;H35I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;T37W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/deletion;P59S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;K60M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;F61P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/deletion;A62R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/deletion;V63A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;D83C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/deletion;V84A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;S85T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/deletion;A86R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/deletion;Y89V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/deletion;H90I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;I/L91K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/deletion;P177S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;F178P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;L179K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;L180K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;A181R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;Q183E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;F183P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;T184W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;Y203V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/ deletion;M204F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;L235K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N236D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;T237W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;P237S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;N238D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H238I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;A238R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;S239T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/ deletion;Q238E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;K239M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/ deletion;L247K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N248D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H248I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;F249P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;M250F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;G251H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/ deletion; andV251A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/ deletion.

Accordingly, another aspect of the present invention contemplates amethod for determining whether an HBV strain exhibits reducedsensitivity to a nucleoside or nucleotide analog or other potentialanti-HBV agent, said method comprising isolating DNA or correspondingmRNA from said HBV and screening for a mutation in the nucleotidesequence of the envelope genes or DNA polymerase gene selected from, inone embodiment, rtS21A, rtL122F, rtN124H, rtH126R, rtT28N, rtP130Q,rtD131N and rtY135C; in another embodiment, rt/N/S/T/I/V53D, rtY126Q,rtL180M, rtS202G, rtI204V and rtI235I/M; in a further embodiment,rtN53D, rtY54H, rtS57P, rtL91I, rtS116P, rtF122L, rtY124H, rtV134D,rtY141Y/F, rtL145M, rtF151F/Y, rtA181T, rtK212R, rtL217R, rtS219A,rtN236T and rtN238D; in yet another embodiment, rtS78T, rtV84M, rtY126C,rtV191I, rtM204I and rtV214A; in still another embodiment rtH90D andrtL/F108L; in even yet another embodiment, rtL157L/M, rtA181V andrtV207I; in even still another embodiment, rtL80V, rtP109S, rtI163V,rtL229M and rtN/H/A/S/Q238K; in another embodiment, rtS78S/T, rtN118N/S,rtN139N/K, rtV142E, rtA181A/T, rtI204M, rtQ/P/S/Stop215Q, rtE218K/E andrtN238N/H; in a further embodiment, sP120T, sM125T and sT127A; in yetanother embodiment, sT118R, sM133T, SF134V, s1195M, sS207R and sY225Y/C;in still another embodiment, sS126T, sM133L/M, sS143S/T, sD144A, sG145Aand sW172Stop; in even yet another embodiment, sN40S, sC69Stop, sM75I,sL88P, sT118A, sW182STOP, sW196L, sY206H and sY225F; in even stillanother embodiment, s181M and sP214Q; in another embodiment, sF83S,sL173F and sW199L; in a further embodiment, sI126T, sK160R, sS174N,sA184V, sW196L, sS210N, sF/C220L and sY221C; in yet another embodiment,sC69Stop/C, sC76Y sI110V/I, sY134N, sW172Stop/W, sW196Stop and sS207R;in still another embodiment, rtK32, rtN33, rtP34, rtH35 and rtT37; ineven yet another embodiment, rtP59, rtK60, rtF61, rtA62 and rtV63; ineven still another embodiment, rtD83, rtV84, rtS85, rtA86, rtY89, rtH90and rtI/L91; in another embodiment, rtP177, rtF178, rtL179, rtL180,rtA181, rtQ182, rtF183 and rtT184; in a further embodiment, rtM204 andrtY203; in yet another embodiment, rt235, rt236, rt237, rt238 and rt239in still another embodiment, rt247, rt248, rt249, rt250 and 11251; andin even yet another embodiment,

K32M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;N33D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/deletion;P34S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;H35I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;T37W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/deletion;P59S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;K60M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;F61P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/deletion;A62R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/deletion;V63A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;D83C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/deletion;V84A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;S85T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/deletion;A86R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/deletion;Y89V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/deletion;H90I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;I/L91K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/deletion;P177S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;F178P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;L179K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;L180K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;A181R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;Q183E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;F183P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;T184W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;Y203V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/ deletion;M204F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;L235K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N236D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;T237W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;P237S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;N238D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H238I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;A238R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;S239T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/ deletion;Q238E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C;K239M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L;L247K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I;N248D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R;H248I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G;F249P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M;M250F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K;G251H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/QE; andV251A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletionor combinations thereof or an equivalent one or more other mutation isindicative of a variant wherein said variant exhibits a decreasedsensitivity to ADV, LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMVand TFV, FTC and ADV, FTC and TFV, FTC and LMV, or ADV and LMV and TFV,or ADV and FTC and TFV, TFV and FTC and LMV, ADV and LMV and FTC, or ADVand FTC and LMV and TFV and/or optionally other nucleoside or nucleotideanalogs or other anti-HBV agents or combination thereof.

The detection of amino acid variants of DNA polymerase is convenientlyaccomplished by reference to the amino acid sequence shown in Formulae Iand II. The polymorphisms shown represent the variations shown invarious databases for active pathogenic HBV strains. Where an HBVvariant comprises an amino acid different to what is represented, thensuch an isolate is considered a putative HBV variant having an alteredDNA polymerase activity.

The present invention further contemplates agents which inhibit ADV,LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV,FTC and TFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTC andTFV, TFV and FTC and LMV, ADV and LMV and FTC, or ADV and FTC and LMVand TFV resistant HBV variants. Such agents are particularly useful iflong term treatment by ADV, LMV, FTC and/or TFV and/or optionally othernucleoside analogs or nucleotide analogs such as TFV is contemplated bythe clinician. The agents may be DNA or RNA or proteinaceous ornon-proteinaceous chemical molecules. Natural product screening such asfrom plants, coral and microorganisms is also contemplated as a usefulpotential source of masking agents as is the screening of combinatorialor chemical libraries. The agents may be in isolated form or in the formof a pharmaceutical composition or formulation and may be administeredin place of or sequentially or simultaneously with a nucleoside ornucleotide analog. Furthermore, rationale drug design is contemplatedincluding solving the crystal or NMR structure of, for example, HBV DNApolymerase and designing agents which can bind to the enzyme's activesite. This approach may also be adapted to other HBV components.

Accordingly, another aspect of the present invention contemplates amethod for detecting an agent which exhibits inhibitory activity to anHBV which exhibits resistance or decreased sensitivity to ADV, LMV, TFV,or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV, FTC andTFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTC and TFV, TFVand FTC and LMV, ADV and LMV and FTC, or ADV and FTC and LMV and TFVand/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combination thereof, said method comprising:

-   -   generating a genetic construct comprising a replication        competent-effective amount of the genome from said HBV contained        in a plasmid vector and then transfecting said cells with said        construct;    -   contacting said cells, before, during and/or after transfection,        with the agent to be tested;    -   culturing said cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to said agent; and    -   subjecting the cells, cell lysates or culture supernatant fluid        to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of said agent.

Still another aspect of the present invention provides a method fordetecting an agent which exhibits inhibitory activity to an HBV whichexhibits resistance or decreased sensitivity to ADV, LMV, TFV, or FTC,or ADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV, FTC and TFV, FTCand LMV, or ADV and LMV and TFV, or ADV and FTC and TFV, TFV and FTC andLMV, ADV and LMV and FTC, or ADV and FTC and LMV and TFV and/oroptionally other nucleoside or nucleotide analogs or other anti-HBVagents or combination thereof, said method comprising:

-   -   generating a genetic construct comprising a replication        competent-effective amount of the genome from said HBV contained        in or fused to an amount of a baculovirus genome effective to        infect cells and then infecting said cells with said construct;    -   contacting said cells, before, during and/or after infection,        with the agent to be tested;    -   culturing said cells for a time and under conditions sufficient        for the HBV to replicate, express genetic sequences and/or        assemble and/or release virus or virus-like particles if        resistant to said agent; and    -   subjecting the cells, cell lysates or culture supernatant fluid        to viral- or viral-component-detection means to determine        whether or not the virus has replicated, expressed genetic        material and/or assembled and/or been released in the presence        of said agent.

Preferably, the HBV genome is stably integrated into the cells' genome.

Particularly useful cells are 2.2.15 cells (Price et al., Proc. Natl.Acad. Sci, USA 86(21): 8541-8544, 1989 or AD cells (also known asHepAD32 cells or HepAD79 cells [Ying et al., Viral Hepat. 7(2): 161-165,2000.

Whilst the baculovirus vector is a particularly useful in the practiceof the present invention, the subject invention extends to a range ofother vectors such as but not limited to adenoviral vectors.

The present invention further extends to cell lines (e.g. 2.2.15 or ADcells) carrying genetic constructs comprising all or a portion of an HBVgenome or a gene or part of a gene therefrom.

The present invention also provides for the use of the subject HBVvariants to screen for anti-viral agents. These anti-viral agentsinhibit the virus. The term “inhibit” includes antagonizing or otherwisepreventing infection, replication, assembly and/or release or anyintermediate step. Preferred anti-viral agents include nucleoside ornucleotide analogs or anti-HBV agents, however, the present inventionextends to non-nucleoside molecules.

In addition, rational drug design is also contemplated to identify orgenerate chemical molecules which either mimic a nucleoside or whichinteract with a particular nucleotide sequence or a particularnucleotide. Combinatorial chemistry and two hybrid screening are some ofa number of techniques which can be employed to identify potentialtherapeutic or diagnostic agents.

In one example, the crystal structure or the NMR structure of polymeraseor the surface antigen is used to rationally design small chemicalmolecules likely to interact with key regions of the molecule requiredfor function and/or antigenicity. Such agents may be useful asinhibitors of polymerase activity and/or may alter an epitope on thesurface antigen.

Several models of the HBV polymerase have been prepared due to thesimilarity with reverse transcriptase from HIV (Das et al., J. Virol.75(10): 4771-4779, 2001; Bartholomeusz et al., Intervirology 40(5-6):337-342 1997; Allen et al., Hepatology 27(6): 1670-1677, 1998). Themodels of the HBV polymerase can be used for the rational drug design ofnew agents effective against HBV encoding the resistant mutations aswell as wild type virus. The rational drug that is designed may be basedon a modification of an existing antiviral agent such as the agent usedin the selection of the HBV encoding the mutations associated withresistance. Viruses or clones expressing HBV genomic material encodingthe mutations may also be used to screen for new antiviral agents.

In an alternative embodiment, the present invention also contemplates amethod for detecting an agent which exhibits inhibitory activity to anHBV polymerase in an in vitro polymerase assay. The HBV polymeraseactivity can be examined using established assays (Gaillard et al.,Antimicrob Agents Chemother. 46(4): 1005-1013, 2002; Xiong et al.,Hepatology 28(6): 1669-1673, 1998).

As indicated above, microarray technology is also a useful means ofidentifying agents which are capable of interacting with defined HBVinternal or external components. For example, arrays of HBV DNApolymerase or peptide fragments thereof carrying different amino acidvariants may be used to screen for agents which are capable of bindingor otherwise interacting with these molecules. This is a convenient wayof determining the differential binding patterns of agents between HBVvariants. Arrays of antibodies may also be used to screen for alteredHBsAg molecules. Microarrays are also useful in proteomic analysis toidentify molecules such as antibodies, interferons or cytokines whichhave an ability to interact with an HBV component. Microarrays of DNAand RNA molecules may also be employed to identify sense and antisensemolecules for genetic regions on the HBV genome or transcripts thereof.

The above methods are particularly useful in identifying an inhibitor ofan HBV resistant to or exhibiting reduced sensitivity to ADV, LMV, TFV,or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV, FTC andTFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTC and TFV, TFVand FTC and LMV, ADV and LMV and FTC, or ADV and FTC and LMV and TFVand/or optionally other nucleoside or nucleotide analogs or otheranti-HBV agents or combination thereof. The present invention extends,therefore, to compositions of the inhibitors. The inhibitors may also bein the form of antibodies or genetic molecules such as ribozymes,antisense molecules and/or sense molecules for co-suppression or theinduction of RNAi or may be other nucleoside or nucleotide analogs orother anti-HBV agents or derivatives of known analogs. Reference to RNAiincludes reference to short, interfering RNAs (siRNA).

The term “composition” includes a “pharmaceutical composition” or aformulation.

The inhibitor is referred to below as an “active ingredient” or “activecompound” and may be selected from the list of inhibitors given above.

The composition may include an antigenic component of the HBV, adefective HBV variant or an agent identified through natural productscreening or rational drug design (including combinatorial chemistry).

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient; use thereof in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The pharmaceutical composition may also comprise genetic molecules suchas a vector capable of transfecting target cells where the vectorcarries a nucleic acid molecule capable of encoding an aspartyl proteaseinhibitor. The vector may, for example, be a viral vector.

Pharmaceutical forms suitable for injectable use include sterile aqueoussolutions (where water soluble) and sterile powders for theextemporaneous preparation of sterile injectable solutions. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dilution mediumcomprising, for example, water, ethanol, polyol (for example, glycerol,propylene glycol and liquid polyethylene glycol, and the like), suitablemixtures thereof and vegetable oils. The proper fluidity can bemaintained, for example, by the use of superfactants. The preventions ofthe action of microorganisms can be brought about by variousanti-bacterial and anti-fungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminium monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with theactive ingredient and optionally other active ingredients as required,followed by filtered sterilization or other appropriate means ofsterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, suitable methods of preparation includevacuum drying and the freeze-drying technique which yield a powder ofactive ingredient plus any additionally desired ingredient.

When the active ingredient is suitably protected, it may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets. For oral therapeuticadministration, the active ingredient may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers and the like.Such compositions and preparations should contain at least 1% by weightof active compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 5 toabout 80% of the weight of the unit. The amount of active compound insuch therapeutically useful compositions is such that a suitable dosagewill be obtained. Preferred compositions or preparations according tothe present invention are prepared so that an oral dosage unit formcontains between about 0.1 μg and 200 mg of active compound. Alternativedosage amounts include from about 1 μg to about 1000 mg and from about10 μg to about 500 mg. These dosages may be per individual or per kgbody weight. Administration may be per hour, day, week, month or year.

The tablets, troches, pills, capsules and the like may also contain thecomponents as listed hereafter. A binder such as gum, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, lactose or saccharin may be added or a flavouringagent such as peppermint, oil of wintergreen or cherry flavouring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills or capsules may be coatedwith shellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and a flavouring. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompound(s) may be incorporated into sustained-release preparations andformulations.

As stated above, the present invention further extends to an isolatedHBsAg from the HBV variants herein described. More particularly, thepresent invention provides an HBsAg or a recombinant form thereof orderivative or chemical equivalent thereof. The isolated surfacecomponent and, more particularly, isolated surface antigen or itsrecombinant, derivative or chemical equivalents are useful in thedevelopment of biological compositions such as vaccine formulations.

Yet another aspect of the present invention provides a compositioncomprising a variant HBV resistant to ADV, LMV, TFV, or FTC, or ADV andLMV, ADV and TFV, LMV and TFV, FTC and ADV, FTC and TFV, FTC and LMV, orADV and LMV and TFV, or ADV and FTC and TFV, TFV and FTC and LMV, ADVand LMV and FTC, or ADV and FTC and LMV and TFV and/or optionally othernucleoside or nucleotide analogs or other anti-HBV agents or an HBVsurface antigen from said variant HBV or a recombinant or derivativeform thereof or its chemical equivalent and one or more pharmaceuticallyacceptable carriers and/or diluents. Such a composition may be regardedas a therapeutic composition and is useful in generating an immuneresponse including a humoral response. Generally, the HBV variants are“defective” and in themselves are unable to cause a sustained infectionin a subject.

As indicated above, antibodies may be generated to the mutant HBV agentsand used for passive or direct vaccination against infection by theseviruses. The antibodies may be generated in humans or non-human animals.In the case of the latter, the non-human antibodies may need to bedeimmunized or more specifically humanized prior to use. Deimmunized mayinclude, for example, grafting complementarity determining regions(CDRs) from the variable region of a murine or non-human animal anti-HBVantibody onto a human consensus fragment antibody binding (Fab)polypeptide. Alternatively, amino acids defining epitopes in thevariable region of the antibody may be mutated so that the epitopes areno longer recognized by the human MHC H complex.

Insofar as ribozyme, antisense or co-suppression (RNAi) or siRNA orcomplexes thereof repression is concerned, this is conveniently aimed atpost-transcription gene silencing. DNA or RNA may be administered or acomplex comprising RNAi or a chemical analog thereof specific for HBVmRNA may be employed.

All such molecules may be incorporated into pharmaceutical compositions.

In another embodiment, the present invention provides a biologicalcomposition comprising a variant HBV or an HBsAg or L, M or S proteinsfrom said variant HBV or a recombinant or derivative form thereof or itschemical equivalent.

Generally, if an HBV is used, it is first attenuated. The biologicalcomposition according to this aspect of the present invention generallyfurther comprises one or more pharmaceutically acceptable carriersand/or diluents.

The biological composition may comprise HBsAg or like molecule from oneHBV variant or the composition may be a cocktail of HbsAgs or L, M or Sproteins or like molecules from a range of ADV- and/or LMV- and/or, FTC-and/or TFV-resistant HBV variants. Similar inclusions apply where thecomposition comprises an HBV.

The present invention is further directed to the use of defective HBVvariants in the manufacture of therapeutic vaccines to vaccinateindividuals against infection by HBV strains having a particularnucleotide sequence or encoding a particular polymerase or surfaceantigen or L, M or S proteins.

Examples of suitable vaccine candidates are defective forms of HBVvariants comprising a mutation selected from, in one embodiment, rtS21A,rtL122F, rtN124H, rtH126R, rtT28N, rtP130Q, rtD131N and rtY135C; inanother embodiment, rt/N/S/T/I/V53D, rtY126Q, rtL180M, rtS202G, rtI204Vand rtI235I/M; in a further embodiment, rtN53D, rtY54H, rtS57P, rtL91I,rtS116P, rtF122L, rtY1241-1, rtV134D, rtY141Y/F, rtL145M, rtF151F/Y,rtA181T, rtK212R, rtL217R, rtS219A, rtN236T and rtN238D; in yet anotherembodiment, rtS78T, rtV84M, rtY126C, rtV191I, rtM2041 and rtV214A; instill another embodiment rtH90D and rtL/F108L; in even yet anotherembodiment, rtL157L/M, rtA181V and rtV207I; in even still anotherembodiment, rtL80V, rtP109S, rtI163V, rtL229M and rtN/H/A/S/Q238K; inanother embodiment, rtS78S/T, rtN118N/S, rtN139N/K, rtV142E, rtA181A/T,rtI204M, rtQ/P/S/Stop215Q, rtE218K/E and rtN238N/H; in a furtherembodiment, sP120T, sM125T and sT127A; in yet another embodiment,sT118R, sM133T, SF134V, sI195M, sS207R and sY225Y/C; in still anotherembodiment, sS126T, sM133L/M, sS143S/T, sD144A, sG145A and sW172Stop; ineven yet another embodiment, sN40S, sC69Stop, sM75I, sL88P, sT118A,sW182STOP, sW196L, sY206H and sY225F; in even still another embodiment,s181M and sP214Q; in another embodiment, sF83S, sL173F and sW199L; in afurther embodiment, sI126T, sK160R, sS174N, sA184V, sW196L, sS210N,sF/C220L and sY221C; in yet another embodiment, sC69Stop/C, sC76YsI110V/I, sY134N, sW172Stop/W, sW196Stop and sS207R; in still anotherembodiment, rtK32, rtN33, rtP34, rtH35 and rtT37; in even yet anotherembodiment, rtP59, rtK60, rtF61, rtA62 and rtV63; in even still anotherembodiment, rtD83, rtV84, rtS85, rtA86, rtY89, rtH90 and rtI/L91; inanother embodiment, rtP177, rtF178, rtL179, rtL180, rtA181, rtQ182,rtF183 and rtT184; in a further embodiment, rtM204 and rtY203; in yetanother embodiment, rt235, rt236, rt237, rt238 and rt239 in stillanother embodiment, rt247, rt248, rt249, rt250 and rt251; and in evenyet another embodiment,

K32M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;N33D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/deletion;P34S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;H35I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;T37W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/deletion;P59S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;K60M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;F61P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/deletion;A62R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/deletion;V63A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;D83C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/deletion;V84A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;S85T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/deletion;A86R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/deletion;Y89V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/deletion;H90I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;I/L91K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/deletion;P177S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;F178P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;L179K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;L180K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;A181R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;Q183E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;F183P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;T184W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;Y203V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/ deletion;M204F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;L235K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N236D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;T237W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;P237S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;N238D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H238I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;A238R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;S239T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/ deletion;Q238E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;K239M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/ deletion;L247K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N248D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H248I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;F249P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;M250F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;G251H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E; andV251A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletionor a combination of two or more mutations.

In one embodiment, for example, an HBV variant may be identified havinga particular mutation in its polymerase conferring resistance ordecreased sensitivity to a nucleoside analog. This variant may then bemutated to render it defective, i.e. attenuated or unable to causeinfection. Such a defective, nucleoside analog-resistant virus may thenbe used as a therapeutic vaccine against virulent viruses having thesame mutation in its polymerase.

The subject invention extends to kits for assays for variant HBVresistant to ADV, LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMV andTFV, FTC and ADV, FTC and TFV, FTC and LMV, or ADV and LMV and TFV, orADV and FTC and TFV, TFV and FTC and LMV ADV and LMV and FTC, or ADV andFTC and LMV and TFV. Such kits may, for example, contain the reagentsfrom PCR or other nucleic acid hybridization technology or reagents forimmunologically based detection techniques. A particularly useful assayincludes the reagents and components required for immobilizedoligonucleotide- or oligopeptide-mediated detection systems.

Still another aspect of the present invention contemplates a method fordetermining the potential for an HBV to exhibit reduced sensitivity toADV, LMV, TFV, or FTC, or ADV and LMV, ADV and TFV, LMV and TFV, FTC andADV, FTC and TFV, FTC and LMV, or ADV and LMV and TFV, or ADV and FTCand TFV, TFV and FTC and LMV, ADV and LMV and FTC, or ADV and FTC andLMV and TFV and/or optionally other nucleoside or nucleotide analogs orother anti-HBV agents or combination thereof, said method comprisingisolating DNA or corresponding mRNA from said HBV and screening for amutation in the nucleotide sequence encoding HBV DNA polymeraseresulting in at least one amino acid substitution, deletion and/oraddition in any one or more of domains F and G, and domains A through toE or a region proximal thereto of said DNA polymerase and associatedwith resistance or decreased sensitivity to ADV, LMV, TFV, or FTC, orADV and LMV, ADV and TFV, LMV and TFV, FTC and ADV, FTC and TFV, FTC andLMV, or ADV and LMV and TFV, or ADV and FTC and TFV, TFV and FTC andLMV, ADV and LMV and FTC, or ADV and FTC and LMV and TFV, wherein thepresence of such a mutation is an indication of the likelihood ofresistance to said ADV, LMV, TFV, or FTC, or ADV and LMV, ADV and TFV,LMV and TFV, FTC and ADV, FTC and TFV, FTC and LMV, or ADV and LMV andTFV, or ADV and FTC and TFV, TFV and FTC and LMV, ADV and LMV and FTC,or ADV and FTC and LMV and TFV.

An assessment of a potential viral variant is important for selection ofan appropriate therapeutic protocol. Such an assessment is suitablyfacilitated with the assistance of a computer programmed with software,which inter alia adds index values (I_(V)s) for at least two featuresassociated with the viral variants to provide a potency value (P_(A))corresponding to the resistance or sensitivity of a viral variant to aparticular chemical compound or immunological agent. The I_(V)s can beselected from (a) the ability to exhibit resistance for reducedsensitivity to a particular compound or immunological agent; (b) analtered DNA polymerase from wild-type HBV; (c) an altered surfaceantigen from wild-type HBV; or (d) morbidity or recovery potential of apatient. Thus, in accordance with the present invention, I_(V)s for suchfeatures are stored in a machine-readable storage medium, which iscapable of processing the data to provide a P_(A) for a particular viralvariant or a biological specimen comprising same.

Thus, in another aspect, the invention contemplates a computer programproduct for assessing the likely usefulness of a viral variant orbiological sample comprising same for determining an appropriatetherapeutic protocol in a subject, said product comprising:

-   (1) code that receives as input I_(V)s for at least two features    associated with said viral agents or biological sample comprising    same, wherein said features are selected from:    -   (a) the ability to exhibit resistance for reduced sensitivity to        a particular compound or immunological agent;    -   (b) an altered DNA polymerase from wild-type HBV;    -   (c) an altered surface antigen from wild-type HBV;    -   (d) morbidity or recovery potential of a patient; or    -   (e) altered replication capacity (increased or decreased);-   (2) code that adds said I_(V)s to provide a sum corresponding to a    P_(V) for said viral variants or biological samples; and-   (3) a computer readable medium that stores the codes.

In a related aspect, the invention extends to a computer for assessingthe likely usefulness of a viral variant or biological sample comprisingsame in a subject, wherein said computer comprises:

-   (1) a machine-readable data storage medium comprising a data storage    material encoded with machine-readable data, wherein said    machine-readable data comprise I_(V)s for at least two features    associated with said viral variant or biological sample; wherein    said features are selected from:—    -   (a) the ability to exhibit resistance for reduced sensitivity to        a particular compound or immunological agent;    -   (b) an altered DNA polymerase from wild-type HBV;    -   (c) an altered surface antigen from wild-type HBV;    -   (d) morbidity or recovery potential of a patient; or    -   (e) altered replication capacity (increased or decreased);-   (2) a working memory for storing instructions for processing said    machine-readable data;-   (3) a central-processing unit coupled to said working memory and to    said machine-readable data storage medium, for processing said    machine readable data to provide a sum of said I_(V)s corresponding    to a P_(V) for said compound(s); and-   (4) an output hardware coupled to said central processing unit, for    receiving said P_(V).

Any general or special purpose computer system is contemplated by thepresent invention and includes a processor in electrical communicationwith both a memory and at least one input/output device, such as aterminal. FIG. 19 shows a generally suitable computer system. Such asystem may include, but is not limited, to personal computers,workstations or mainframes. The processor may be a general purposeprocessor or microprocessor or a specialized processor executingprograms located in RAM memory. The programs may be placed in RAM from astorage device, such as a disk or pre-programmed ROM memory. The RAMmemory in one embodiment is used both for data storage and programexecution. The computer system also embraces systems where the processorand memory reside in different physical entities but which are inelectrical communication by means of a network.

In an alternative embodiment, the program screens for a mutationselected from, in one embodiment, rtS21A, rtL122F, rtN124H, rtH126R,rtT28N, rtP130Q, rtD131N and rtY135C; in another embodiment,rt/N/S/T/I/V53D, rtY126Q, rtL180M, rtS202G, rtI204V and rtI235I/M; in afurther embodiment, rtN53D, rtY54H, rtS57P, rtL91I, rtS116P, rtF122L,rtY124H, rtV134D, rtY141Y/F, rtL145M, rtF151F/Y, r/A181T, rtK212R,rtL217R, rtS219A, rtN236T and rtN238D; in yet another embodiment,rtS78T, rtV84M, rtY126C, rtV191I, rtM204I and rtV214A; in still anotherembodiment rtH90D and rtL/F108L; in even yet another embodiment,rtL157L/M, rtA181V and rtV207I; in even still another embodiment,rtL80V, rtP109S, rt1163V, rtL229M and rtN/H/A/S/Q238K; in anotherembodiment, rtS78S/T, rtN118N/S; rtN139N/K, rtV142E, rtA181A/T, rt1204M,rtQ/P/S/Stop215Q, rtE218K/E and rtN238N/H; in a further embodiment,sP120T, sM125T and sT127A; in yet another embodiment, sT118R, sM133T,SF134V, sI195M, sS207R and sY225Y/C; in still another embodiment,sS126T, sM133L/M, sS143S/T, sD144A, sG145A and sW172Stop; in even yetanother embodiment, sN40S, sC69Stop, sM75I, sL88P, sT118A, sW182STOP,sW196L, sY206H and sY225F; in even still another embodiment, s181M andsP214Q; in another embodiment, sF83S, sL173F and SW199L; in a furtherembodiment, sI126T, sK160R, sS174N, sA184V, sW196L, sS210N, sF/C220L andsY221C; in yet another embodiment, sC69Stop/C, sC76Y sI110V/I, sY134N,sW172Stop/W, sW196Stop and sS207R; in still another embodiment, rtK32,rtN33, rtP34, rtH35 and rtT37; in even yet another embodiment, rtP59,rtK60, rtF61, rtA62 and rtV63; in even still another embodiment, rtD83,rtV84, rtS85, rtA86, rtY89, rtH90 and rtI/L91; in another embodiment,rtP177, rtF178, rtL179, rtL180, rtA181, rtQ182, rtF183 and rtT184; in afurther embodiment, rtM204 and rtY203; in yet another embodiment, rt235,rt236, rt237, rt23S and rt239 in still another embodiment, rt247, rt248,rt249, rt250 and rt251; and in even yet another embodiment,

K32M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;N33D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/deletion;P34S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;H35I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;T37W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/deletion;P59S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/deletion;K60M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/deletion;F61P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/deletion;A62R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/deletion;V63A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;D83C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/deletion;V84A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletion;S85T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/deletion;A86R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/deletion;Y89V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/deletion;H90I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/deletion;I/L91K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/deletion;P177S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;F178P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;L179K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;L180K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;A181R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;Q183E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;F183P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;T184W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;Y203V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/ deletion;M204F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;L235K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N236D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;T237W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/ deletion;P237S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/ deletion;N238D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H238I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;A238R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/ deletion;S239T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/ deletion;Q238E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/ deletion;K239M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/ deletion;L247K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/ deletion;N248D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/ deletion;H248I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/ deletion;F249P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/ deletion;M250F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/ deletion;G251H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E; andV251A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/deletionor a combination of two or more mutations.

The present invention is further described by the following non-limitingExamples.

Example 1 Overlapping Genome of HBV

The overlapping genome of HBV is represented in FIG. 1. The geneencoding DNA polymerase (P), overlaps the viral envelope genes, Pre-S1and Pre-S2, and partially overlaps the X and core (C) genes. The HBVenvelope comprises small, middle and large proteins HBV surfaceantigens. The large protein component is referred to as the HBV surfaceantigen (HBsAg) and is encoded by the S gene sequence. The Pre-S1 andPre-S2 gene sequences encode the other envelope components.

Example 2 Patients and Treatment

Patient A, a 48 year old Lebanese woman was initially referred forevaluation of thrombocytopenia and hepatosplenomegaly. At this time thepatient had abnormal LFT's (ALT 67 U/L, normal <55) and the HBV DNA was61 pg/ml (231 days prior to the start of treatment). The patient wasHBsAg and HBeAg positive. The ALT's fluctuated between 50-70 IU/L from(−231 to −35 days pretreatment). ADV was commenced on Day 0 in aclinical trial on 30 mg/day. HBV DNA levels were reduced with ADVtreatment. The ADV treatment was reduced to 10 mg/day (144 dayspost-treatment). There was a problem with the randomization treatmentprotocol. The patient was on antiviral treatment for 1 month only duringthe second year of the treatment period. The study was completed on Day679 post ADV treatment. The patient was not on ADV treatment until theopen label ADV was recommenced on Day 875 from the start of the initialADV treatment. This second period of ADV treatment was given for 108days (day 983 post initial ADV treatment). The HBV DNA levels remainedat 7-10 pg/ml (1.96×10⁵ to 2.8×10⁵ copies/ml). At Day 983, ADV treatmentwas stopped and the patient was treated with LMV.

Patient B is a male liver transplant patient. The patient has been onboth sequential and combination antiviral therapy including HBIG,FCV+HBIG, LMV+HBIG, LMV, LMV+GCV, LMV+FCV+GCV, LMV+GCV and finallyLMV+ADV. The patient has been on long term ADV+LMV treatment for over795 days.

Patient C, is a 58 year old male. Prior to ADV treatment the patient hadabnormal LFT's (ALT 240 IU/L, normal <55) and the HBV DNA was 2×10⁷copies/ml. ADV was commenced on Day 0 in a clinical trial on 10 mg/dayfor two years. The average ALT during the two year clinical trial periodws 114 IU/L. However, the ALT was rising and at 630 days after the startof ADV treatment the ALT remained high 407 IU/L. Open label ADV wascommenced on Day 668 from the start of the initial ADV treatment. Thissecond period of ADV treatment was given for 71 days. The HBV DNA levelsremained high during open label ADV treatment (3.7×10⁶ to 1.5×10⁷copies/ml). The peak ALT during open label ADV treatment was 517 IU/L(Day 738). The next day (Day 739), ADV treatment was stopped and thepatient was treated with LMV.

Example 3 Detection of Viral Markers

Hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg),anti-HBe and hepatitis B core antigen (HBcAg) specific IgG and IgM weremeasured using commercially available immunoassays (Abbott Laboratories,North Chicago, Ill., USA). Hepatitis B viral DNA levels were measuredusing a capture hybridization assay according to the manufacturer'sdirections (Digene Hybrid Capture II, Digene Diagnostics Inc.,Beltsville, Md.). The manufacturers stated cut-off for detecting HBVviremia in clinical specimens was 0.7×10⁶ copies/ml or 2.5 pg/ml,[Hendricks et al., Am J Clin Pathol 104: 537-46, 1995]. HBV DNA levelscan also be quantitated using other commercial kits such as Cobasamplification HBV monitor kit (Roche).

Example 4 Sequencing of HBV DNA

HBV DNA was extracted from 100 μl of serum as described previously byAye et al., J. Hepatol. 26: 1148-1153, 1997. Oligonucleotides weresynthesized by Geneworks, Adelaide, Australia. Amplification of the HBVpolymerase gene has been described by Aye et al., 1997, supra.

The specific amplified products were purified using PCR purificationcolumns from MO BIO Laboratories Inc (La Jolla, Calif.) and directlysequenced using Big Dye terminator Cycle sequencing Ready Reaction Kit(Perkin Elmer, Cetus Norwalk, Conn.). The PCR primers were used assequencing primers, OS1 5′-GCC TCA TTT TGT GGG TCA CCA TA-3′ (nt1408-1430) [SEQ ID NO:3], TTA3 5′-AAA. TTC GCA GTC CCCAAA-3′(nt2128-2145) [SEQ ID NO:4], JM 5′-TTG GGG TGG AGC CCT CAGGCT-3′(nt1676-1696) [SEQ ID NO:5], TTA4 5′-GAA AAT TGG TAA CAG CGG-3′(nt 2615-2632) [SEQ ID NO:6], OS2 5′ TCT CTG ACA TAC TTT CCA AT 3′ (nt2798-2817) [SEQ ID NO:7], to sequence the internal regions of the PCRproducts.

Example 5 Analysis of HBV DNA

Patient A: During ADV treatment, unique HBV mutations were detected bysequencing (Tables 4 and 5) This includes the unique mutation at rtY135Cin addition to the mutation at rtT128N that was present prior to ADVtreatment. A number of other unique changes were also detected in thepolymerase and in the overlapping envelope gene (Table 5, FIGS. 4, 5 and6). The unique change in the HBsAg include sP120T. These unique changeswere compared to reference sequences from each of the seven genotypesA-G as well as a consensus sequence from pretreatment samples todetermine unique changes.

Patient B: The HBV mutations prior to ADV treatment and during ADVtreatment are listed in Table 6 and 7 and FIGS. 7, 8, and 9. The uniquechanges in the rt region of the HBV DNA polymerase includertN/S/T/I/V53D, rtY126Q, rtL180M, rtS202G, rt1204V and rtI235I/M. Theunique changes in the HBsAg include sT118R, sM133T, sF134V, sI195M,sS207R, sY225Y/C.

Patient C: The HBV mutations prior to ADV treatment and during ADVtreatment are listed in Tables 8 and 9 and FIGS. 10, 11 and 12. Theunique changes in the rt region of the HBV DNA polymerase includertN53D, rtS116P, rtF151F/T, rtN236T and rtN238D. The unique changes inthe HBsAg include sG145A and sW172stop.

Patient D: The HBV mutations during ADV treatment is listed in Table 10and FIGS. 13, 14 and 15. The unique changes in the HBV DNA polymeraseinclude rtS78T, rtV84M, rtY126C, rtV191I, rtM2041 and rtV214A. Theunique changes in the surface include sN40S and sC69 Stop. A number ofunique changes were detected after the stop codon mutation at codon 69of the S gene including sM75I, sL88P, sT118A, sW182stop, sW196L, sY206Hand sY225F.

Patient E: The HBV mutations during ADV treatment is listed in Table 11and FIGS. 16, 17 and 18. The unique changes in the HBV DNA polymeraseinclude rtH90D and rtL/F108L. The unique changes in the surface includesI81M and sP214Q. A six nucleotide insertion was also detected resultingin a two amino acid insertion in the HBV polymerase and envelope gene atcodons rt131 and s122, respectively. This insertion was previouslydetected in pre-ADV samples.

Example 6 Adefovir Dipivoxil (ADV)

ADV (formerly Bis-pom PMEA)) is a potent inhibitor of HBV replication.The structure of ADV is shown in FIG. 2 and its synthesis is describedby Benzaria et al., J Med. Chem. 39: 4958-4965, 1996).

Example 7 HBV rt Mutants

The HBV polymerase has similarities to other polymerases including HIV.Thus, mutations associated with resistance to antiviral agents may occurwithin the polymerase in functionally important regions such as thenucleotide triphosphate binding pocket that may also include theinteraction between the DNA primer and template strand, magnesium ionsand nucleoside triphosphates or nucleoside/nucleotide analogs (and therevarious phosphroylated forms). Codons which are proposed to be mutatedduring anti-viral selection pressure are rtK32, rt N33, rtP34, rtH35 andrtT37 (that are upstream from the F domain); rt P59, rtK60, rtF61, rtA62and rtV63 (between the F and A domains), rtD83, rtVS4, rtS85, rtA86, rtY89, rt H90 and rtI/L91 (within the A domain and the region immediatelyprior to and after), rtP177, rtF178, rt L179, rtL180, rtA181, rtQ182,rtF183 and rtT184 (B domain); rtM204 and rtY203(C Domain), rtL235,rtN236, rtP/T237, rtN/H/A/S/Q238 and rtK239 (D Domain), rLt247,rtN/H248, rtF249, rtM250 and rtG251 (E Domain). The codons are definedin Table 12 and examples of various mutants are given in Tables 13 and14.

Example 8 Patient F

The HBV mutations during ADV treatment of Patient F are listed in Table15 and FIGS. 20, 21 and 22. The unique changes in the HBV DNA polymeraseincludes rtL157L/M, rtA181V, rtV2071, and rtN236T. The unique changes inthe surface includes sF83S, sL173F and sW199L.

Example 9 Patient G

The HBV mutations during ADV treatment of Patient G are listed in Table16 and FIGS. 23, 24 and 25. The unique changes in the HBV DNA polymeraseincludes rtL80V, rtP109S, rtI163V, rtM2041, rtL229M and rtN/H/A/S/Q238K.The unique changes in the surface includes sI126T, sK160R, sS174N,sA184V, sW196L, sS210N, sF/C220L and sY221C.

Example 10 Patient H

The HBV mutations during ADV treatment in Patient H are listed in Table17 and FIGS. 26, 27 and 28. The unique changes in the HBV DNA polymeraseincludes rtS78S/T, rtN118N/S, rtN139N/K, rtV142E, rtA181A/T, rtI204M,rtQ/P/S/Stop215Q, rtE218K/E, and rtN238N/H. The unique changes in thesurface include sC69Stop/C, sC76Y sI110V/I, sY134N, sW172Stop/W,sW196Stop and sS207R.

Example 11 In Vitro Analysis of ADV Resistance

The sensitivity/resistance profile of HBV mutants to ADV was examined invitro using recombinant HBV/baculovirus. The procedure for analyzing theresistance profile is outlined in the following Examples 12-20.

Example 12 Cell Culture

Sf21 insect cells were maintained in supplemented Grace's insect mediumfurther supplemented with 10% v/v heat-inactivated fetal bovine serum(Gibco BRL, Gaithersburg, Md.) in humidified incubator at 28° C. withCO₂. HepG2 cells were maintained in minimal essential mediumsupplemented with 10% v/v heat-inactivated fetal bovine serum (MEM-FBS).HepG2 cells were grown in humidified 37° C. incubators at 5% v/v CO₂.

Example 13 Preparation of HBV/Baculovirus Transfer Vector with SpecificPoint Mutations

The recombinant HBV/baculovirus system used for antiviral testing hasbeen previously described (Delaney et al., Antimicrob Agents Chemother45(6): 17054013, 2001). In brief, the recombinant transfer vector wascreated by excising a fragment containing the 1.3×HBV genome constructand cloning it into the multiple cloning region of a baculovirus vectorpBlueBac4.5 (Invitrogen, Carlsbad, Calif.). Point mutations were createdby site directed mutagenesis using the commercial kits according to themanufacturer's specifications (QuikChange, Stratagene). HBV/baculovirusrecombinant clones encoding the reverse transcriptase mutationsrtA181T/N236T/N238D and rtN236T/N236D in combination with the precoremutation at G1896A (pcW28 stop) or wild-type with respect to codonpcW28, were prepared by site-directed mutagenesis. The nucleotidesequence of the plasmid and the point mutations generated by sitedirected mutagenesis were confirmed by sequencing using the ABI PrismBig Dye Terminator Cycle Sequencing Ready Reaction Kit according to themanufacturer's specifications (Perkin Elmer, Cetus Norwalk, Conn.).

Example 14 Generation of Recombinant Baculoviruses Containing the 1.3HBV Construct

Purified recombinant transfer vector and linear AcMNPV baculovirus DNAwere co-transfected into Sf21 cells using the BacNBlue transfection kitfrom Invitrogen (Carlsbad, Calif.); recombinant viruses were isolated byplaque assay according to the manufacturer's instructions. A series ofrecombinant viruses were amplified from isolated plaques by infecting100-mm dishes of Sf21 cells. Viral DNA was extracted from amplifiedviruses using standard procedures. Purified viral DNA was digested withrestriction enzymes and then fractionated by electrophoresis in a 1% v/vagarose gel. Southern blotting was performed to deteiuiine which virusisolates contained the intact 1.3 HBV construct. A Boehringer MannheimRandom Prime DNA Labeling kit (Indianapolis, Ind.) was used to generate[P³²]-radiolabeled probes. A full-length double-stranded HBV genome wasused as a template for all radiolabeled probes. Viral DNA sequence wasconfirmed by PCR amplification of the polymerase catalytic region usingthe sense primer 5′-GCC TCA TTT TGT GGG TCA CCA TA-3′ [SEQ ID NO:8],(nucleotide 1408 to 1430 according to HBV Genebank Accession numberM38454) and the antisense primer 5′-TCT CTG ACA TAC TTT CCA AT-3′ [SEQID NO:9] (nucleotides 2817 to 2798 according to HBV Genebank Accessionnumber M38454). The following primers were utilized for the sequencingof internal regions 5′-TGC ACG ATT CCT GCT CAA-3′ [SEQ ID NO:10](nucleotides 2345-2362 according to HBV Genebank Accession numberM38454) and 5′-TTT CTC AAA GGT GGA GAC AG-3′ [SEQ ID NO:11] (nucleotides1790-1810 according to HBV Genebank Accession number M38454).

Example 15 Preparative Baculovirus Amplification and Purification

Baculoviruses were amplified by infecting suspension cultures of Sf21cells in log phase at a multiplicity of infection (moi) of 0.5 pfu/cell.Infections were allowed to proceed until a majority of the cells in theflasks showed visible signs of infection (four to five days). Virionswere concentrated from infected Sf21 medium by centrifugation at80,000×g and purified through a 20-60% w/v sucrose gradient. Purifiedvirus was titrated in quadruplicate in Sf21 cells by end-point dilution.An aliquot of each high titer stock was used for DNA extraction. Thepolymerase gene was amplified and sequenced to confirm the presence ofthe site-directed mutagenesis as in Example 14.

Example 16 Infection of HepG2 Cells with Recombinant HBV ExpressingBaculovirus

HepG2 cells were seeded at approximately 20-40% confluency and then weregrown for 16-24 hours before infection. On the day of infection,triplicate plates of cells were trypsinized, and viable cell number wasdetermined with a hemocytometer using Trypan blue exclusion. Averagecell counts were calculated and used to determine the volume ofhigh-titer viral stock necessary to infect cells at the indicated moi.HepG2 cells were washed one time with serum-free MEM to remove traces ofserum. Baculovirus was diluted into MEM without serum to achieve theappropriate moi using volumes of 1.0, 0.5, and 0.25 ml to infect 100-mm,60 mm, and 35-mm dishes, respectively. Baculovirus was adsorbed to HepG2cells for one hour at 37° C. with gentle rocking every 15 minutes toensure that the inoculum was evenly distributed. The inoculum was thenaspirated and HepG2 cells were washed two times with phosphate-bufferedsaline and refed MEM-FBS with or without various concentrations ofagents.

Example 17 Detection of Intracellular Replicative Intermediates

HBV core particles were isolated from the cytoplasmic fraction of HepG2cells lysed in 0.5% w/v NP-40. Cytoplasmic extracts were adjusted to 10mmol/l McC12 and unprotected DNA was removed by an incubation to 500g/ml Proteinase K for 1.5 hours at 37° C. 1113V DNA in the samples werethen extracted using commercial DNA extraction kits such as Qiagen (DNAextraction) or in-house methods using sequential phenol and chloroformextractions, and the nucleic acids were recovered by ethanolprecipitation. Nucleic acids were resuspended in 50 μl/l TE (10 mmol/lTris, 1 mmol/l ethylenediaminetetraacetic acid), normalized by OD260,and digested with 100 g/ml. RNase (Boehringer Mannheim, Indianapolis,Ind.) for one hour at 37° C. before analysis by real-time PCR orelectrophoresis and Southern blotting. After southern blot analysis aBioRad GS-670 imaging densitometer and the Molecular Analyst software(BioRad, Hecules Calif.) was used to analyze suitable exposures ofSouthern blots. Densitometry data was fitted to logistic dose responsecurves using the TableCurve 2D software package from Jandel Scientific.Logistic dose response equations were used to calculate IC₅₀ and IC₉₀values and co-efficients of variation.

Example 18 Real-Time PCR

For the real-time PCR based assay for HBV, HBV DNA was extracted from200 μl of serum using the QIAamp DNA Mini Kit according to themanufacturer's instructions (QIAGEN GmbH, lindens, Germany). Primers anda molecular beacon were designed for conserved nucleic acid sequenceswithin the precore domain of the HBV genome to amplify and detect a216-nucleotide product. Amplification was performed in a 50-μl reactionmixture containing 1.0 Taqman buffer A (Applied Biosystems, Foster City,Calif.), 3.0 mM MgCl, 0.4 pmol of each primer per μL, forward primer,PC1 (5′-GGGAGGAGATTAGGTTAA-3′ [SEQ ID NO:12]) and reverse primer, PC2(5′-GGCAAAAACGAGAGTAACTC-3′ [SEQ ID NO:13]), 0.4 μmol of theHBV-specific molecular beacon per μL,(5′-FAM-CGCGTCCTACTGTTCAAGCCTCCAAGCTGT GACGCG-DABCYL-3′ [SEQ ID NO:14];where FAM represents fluorophore 6-carboxyfluorescein and DABCYL,4-dimethylaminophenylazobenzoic acid, a quenching chromophore) and 1.25U of AmpliTaq Gold DNA polymerase (Perkin-Elmer). PCR was performedusing the ABI PRISM 7700 spectrofluorometric thermocycler (AppliedBiosystems). The PCR program consisted of an initial cycle (95° C. for10 minutes) followed by 45 amplification cycles (94° C. for 15 secs, 50°C. for 30 secs, 72° C. for 30 secs). The instrument detected andrecorded the fluorescence spectrum of each reaction tube during theannealing phase.

An external standard was constructed by ligation of a 1.3 kB wild-typeHBV plasmid (genotype D) into the pBlueBac plasmid vector (HersheyMedical Center, Hershey, Pa.). Quantification of the DNA concentrationof the plasmid was determined by spectrophotometry. Duplicates of serial10-fold dilutions of the plasmid ranging from 108 copies/ml to 100copies/ml were included in each run in order to generate a standardcurve. The copy number in each experimental reaction was determined byinterpolation of the derived threshold cycle (C_(T)).

Example 19 ADV Treatments

ADV was resuspended in sterile water, aliquoted, and frozen at −20° C.to avoid repeated freezing and thawing of the drug. Medium containingADV was prepared daily as needed using fresh aliquots of 3TC. Inexperiments in which ADV treatment was initiated after viral infection,HepG2 cells were exposed to the indicated concentration of ADVimmediately after infection with HBV baculovirus. In experimentsutilizing pretreatment with ADV, cells were fed medium containing ADV 16hours prior to HBV baculovirus infection, HBV baculovirus infection wasalso carried out in medium containing ADV, and cells were refed freshmedium containing ADV immediately after completion of the infection andwashing procedures.

Example 20 Antiviral Testing Performed with Wild-Type andHBV/Baculovirus Encoding rtA181T/N236T/N238D and rtN236T/N236D

The in vitro antiviral drug cross-resistance testing of the HBV mutantsis shown in Table 18. The laboratory reference strain of HBV (genotype Dsubtype ayw) containing the introduced D domain mutations demonstratedincreased IC₅₀ values against ADV (Table 18). The rt N236T/N238Dmutation was associated with a twenty-three fold increase in IC₅₀against ADV. This was reduced to a five-fold increase when the rtA181Twas also present and this triple HBV polymerase mutant was resistant toLMV.

TABLE 4 Clinical, virological and HBV sequencing data summary forPatient A while on open label ADV. Key polymerase Days HBV DNA mutationspost-ADV copies/ml ALT Treatment detected by treatment (pg/ml) IU/Lprotocol sequencing¹ −230 1.7 10⁶ (61) 67 U/L pre-therapy rtT/N128T/NrtQ/H/R215Q/stop 875 ADV recommenced 904 1.55 × 10⁶ 932 2.97 × 10⁶ 9591.76 × 10⁶ 983 1.64 × 10⁶ 65 end ADV rtT128N rtY135C ¹Nomenclatureaccording to Stuyver et al., 2001, supra

TABLE 5 Summary of HBV mutations in patient A treated with ADV DaysSample post-ADV name treatment Genotype Polymerase* Surface ILA1 −230 DrtA/S21A/S sP120P/T rtT/N128T/N** sI208I/L rtQ/H/R215Q/stop ILA2 904 DrtA/S21S sP/T120P rtF122L sT125M rtR126H sI/I208I/L rtT/N128T/N rtQ130PrtN131D rtQstop/215Q rtH248N ILA3 932 D rtA/S21S sP/T120P rtF122L sT125MrtR126H sI/I208I/L rtT/N128T/N rtQ130P rtN131D rtQstop/215Q rtH248N ILA4983 D rtS21A sP120T rtL122F sM125T rtN124H sT127A rtH126R rtT128NrtP130Q rtD131N rtY135C *Nomenclature according to Stuyver et al., 2001,supra. **Mutations in bold have not been detected in reference HBVgenotypes, mutations not in bold are changes from the previous samplethat are present in reference genotypes.

TABLE 6 Clinical, virological and HBV sequencing data summary forPatient B while on open label ADV. Key polymerase Days HBV DN mutationspost-ADV copies/ml ALT Treatment detected by treatment (pg/ml) IU/Lprotocol sequencing¹ −867(S0)  183 298 pre-therapy rtN/S/T/I/V53DrtV153G rtQ/E215S rtN248H −8(S6) 955 427 pre-ADV on rtI/L80L LMV rtY126QrtL180M rtS202G rtI204V 76(S8) not detected 150 on ADV (20 mg)rtN/S/T/I/V53D and LMV rtY126Q rtL180M rtS202G rtI204V 637(S12) notdetected 36 on ADV (5 mg) rtN/S/T/I/V53D and LMV rtY126Q rtL180M rtS202GrtI204V 872(S15) not detected 67 on ADV (5 mg) rtN/S/T/I/V53D and LMVrtY126Q rtL180M rtS202G rtI204V rtI235I/M ¹Nomenclature according toStuyver et al., 2001, supra

TABLE 7 Summary of HBV mutations in Patient B treated with ADV DaysSample post-ADV name treatment Genotype Polymerase* Surface S0 −867 DrtN/S/T/I/V53D sM/K/L133T rtV153G sF134V rtQ/E215S sS207R rtN248HsL21V/L S6 −8 D rtI/L80L sT11SR rtY126Q sM133T rtL180M sF134V rtS202GsI195M rtI204V sS207R S8 76 D rtN/S/T/I/V53D sT11SR rtY126Q sM133TrtL180M sF134V rtS202G sI195M rtI204V sS207R S12 637 D rtN/S/T/I/V53DsT118R rtY126Q sM133T rtL180M sF134V rtS202G sI195M I204V sS207R S15 872D rtN/S/T/I/V53D sT118R rtY126Q sM133T rtL180M sF134V rtS202G sSI195MrtI204V sS207R rtI235I/M sY225Y/C *Nomenclature according to Stuyver etal., 2001, supra **Mutations in bold have not been detected in referenceHBV genotypes, mutations not in bold are changes from the previoussample that are present in reference genotypes.

TABLE 8 Clinical, virological and HBV sequencing data summary forPatient C while on open label ADV. Key polymerase Days HBV DNA mutationspost-ADV copies/ml ALT Treatment detected by treatment (pg/ml) IU/Lprotocol sequencing¹ −26   2 × 10⁷ pre-therapy rtN53D rtS116PrtD/N/S134V rtN238D 0 240 ADV commenced clinical trial 29 160 630 407668 Open label ADV 701 1.5 × 10⁷ 226 730 3.7 × 10⁶ 361 rtN53D rtS116PrtF151S/T rtA181T rtN236T rtN238D 738 517 739 end ADV, start LMV¹Nomenclature according to Stuyver et al., 2001, supra

TABLE 9 Summary of HBV mutations in Patient C treated with ADV DaysSample post-ADV name treatment Genotype Polymerase* Surface DRJ1299 −26D rtN53D** T126S rtY54H S204G rtS57P L209V rtL91I S210R rtS116P rtF122LrtY124H rtD/N/S134V rtK212R rtL217R rtS219A rtN238D DRJ1 730 D rtN53DsS126T rtY54H sM133L/M rtS57P sS143S/T rtL91I sD144A rtS116P sG145ArtF122L sW172Stop rtY124H rtV134D rtY141Y/F rtL145M rtF151T/F rtA181TrtK212R rtL217R rtS219A rtN236T rtN238D *Nomenclature according toStuyver et al., 2001, supra. **Mutations in bold have not been detectedin reference HBV genotypes, mutations not in bold are changes from theprevious sample that are present in reference genotypes.

TABLE 10 Summary of HBV mutations in Patient D treated with ADV SampleName Genotype Polymerase* Surface 02575908 D rtS78T sN40S rtV84MsC69stop rtY126C sM75I rtV191I sL88P rtM204I sT118A rtV214A SW182STOPsW196L sY206H sY225F *Nomenclature according to Stuyver et al., 2001,supra. **Mutations in bold have not been detected in reference HBVgenotypes, mutations not in bold are changes from the previous samplethat are present in reference genotypes.

TABLE 11 Summary of HBV mutations in Patient E treated with ADV SampleName Genotype Polymerase* Surface 8123/02 A rtH90D sI81M rtL/F108LsY/S100Y 6nt insertion/ 6nt insertion/ duplication duplication aftercodon after codon rt131(aaQ&N) s122 (aaT & K) sP214Q *Nomenclatureaccording to Stuyver et al., 2001, supra. **Mutations in bold have notbeen detected in reference HBV genotypes, mutations not in bold arechanges from the previous sample that are present in referencegenotypes.

TABLE 12 Codons where mutations occur following exposure to nucleosideor nucleotide analogs Original amino acid in reverse trans- Region/criptase (rt) and Domain codon position Nucleotide prior to F K32 AAGAAA N33 AAT P34 CCT H35 CAC T37 ACC F TO A P59 CCA K60 AAA F61 TTC A62GCA V63 GTC A D83 GAT V84 GTG S85 TCT A86 GCG Y89 TAT H90 CAT I/L91 ATTCTT B P177 CCG F178 TTT L179 CTC L180 CTG A181 TTG Q182 CAG F183 TTTT184 ACT C Y203 TAT M204 ATG D L235 TTG TTA N236 AAC AAT T237 ACT ACCP237 CCT CCC N238 AAT AAC H238 CAC A238 GCT S238 TCT Q238 CAG K239 AAAAAG E L247 CTT TTA CTA CTC CTG N248 AAC AAT H248 CAT CAC F249 TTC TTTM250 ATG G251 GGT GGA GGC GGG V251 GTC

TABLE 13 Target amino acid sites in rt with codons and mutations leadingto amino acid changes.

Title Codon Amino Acid Codon Amino Acid Codon Amino Acid K32 AAG Lys AAGLys GAG Glu N33 AAT Asn AAT Asn GAT Asp P34 CCT Pro ACT Thr GCT Ala H35CAC His AAC Asn GAC Asp T37 ACC Thr ACC Thr GCC Ala P59 CCA Pro ACA ThrGCA Ala K60 AAA Lys AAA Lys GAA Glu F61 TTC Phe ATC Ile GTC Val A62 GCAAla ACA Thr GCA Ala V63 GTC Val ATC Ile GTC Val D83 GAT Asp AAT Asn GATAsp V84 GTG Val ATG Met GTG Val S85 TCT Ser ACT Thr GCT Ala A86 GCG AlaACG Thr GCG Ala Y89 TAT Tyr AAT Asn GAT As H90 CAT His AAT Asn GAT AspI/L91 ATT Ile ATT Ile GTT Val P177 CCG Pro ACG Thr GCG Ala F178 TTT PheATT Ile GTT Val L179 CTC Leu ATC Ile GTC Val L180 CTG Leu ATG Met GTGVal A181 TTG Leu ATG Met GTG Val Q183 CAG Gln AAG Lys GAG Glu F183 TTTPhe ATT Ile GTT Val T184 ACT Thr ACT Thr GCT Ala Y203 TAT Tyr AAT AsnGAT Asp M204 ATG Met ATG Met GTG Val L235 TTG Leu ATG Met GTG Val N236AAC Asn AAC Asn GAC Asp T237 ACT Thr ACT Thr GCT Ala P237 CCT Pro ACTThr GCT Ala N238 AAT Asn AAT Asn GAT Asp H238 CAC His AAC Asn GAC AspA238 GCT Ala ACT Thr GCT Ala S239 TCT Ser ACT Thr GCT Ala Q238 CAG GlnAAG Lys GAG Glu K239 AAA Lys AAA Lys GAA Glu L247 CTT Leu ATT Ile GTTVal N248 AAC Asn AAC Asn GAC Asp H248 CAT His AAT Asn GAT Asp F249 TTCPhe ATC Ile GTC Val M250 ATG Met ATG Met GTG Val G251 GGT Gly AGT SerGGT Gly V251 GTC Val ATC Ile GTC Val Codon Amino Acid Codon Amino AcidCodon Amino Acid CAG Gln TAG Stop AAG Lys CAT His TAT Tyr AAT Asn CCTPro TCT Ser CAT His CAC His TAC Tyr CAC His CCC Pro TCC Ser AAC Asn CCAPro TCA Ser CAA Gln CAA Gln TAA Stop AAA Lys CTC Leu TTC Phe TAC Tyr CCAPro TCA Ser GAA Glu CTC Leu TTC Phe GAC Asp CAT His TAT Tyr GAT Asp CTGLeu TTG Leu GAG Glu CCT Pro TCT Ser TAT Tyr CCG Pro TCG Ser GAG Glu CATHis TAT Tyr TAT Tyr CAT His TAT Tyr CAT His CTT Leu TTT Phe AAT Asn CCGPro TCG Ser CAG Gln CTT Leu TTT Phe TAT Tyr CTC Leu TTC Phe CAC His CTGLeu TTG Leu CAG Gln CTG Leu TTG Leu TAG Stop CAG Gln TAG Stop CAG GlnCTT Leu TTT Phe TAT Tyr CCT Pro TCT Ser AAT Asn CAT His TAT Tyr TAT TyrCTG Leu TTG Leu AAG Lys CTG Leu TTG Leu TAG Stop CAC His TAC Tyr AAC AsnCCT Pro TCT Ser AAT Asn CCT Pro TCT Ser CAT His CAT His TAT Tyr AAT AsnCAC His TAC Tyr CAC His CCT Pro TCT Ser GAT Asp CCT Pro TCT Ser TAT TyrCAG Gln TAG Stop CAG Gln CAA Gln TAA Stop AAA Lys CTT Leu TTT Phe CATHis CAC His TAC Tyr AAC Asn CAT His TAT Tyr CAT His CTC Leu TTC Phe TACTyr CTG Leu TTG Leu AAG Lys CGT Arg TGT Cys GAT Asp CTC Leu TTC Phe GACAsp Codon Amino Acid Codon Amino Acid Codon Amino Acid AGG Arg ACG ThrATG Met AGT Ser ACT Thr ATT Ile CGT Arg CCT Pro CTT Leu CGC Arg CCC ProCTC Leu AGC Ser ACC Thr ATC Ile CGA Arg CCA Pro CTA Leu AGA Arg ACA ThrATA Ile TGC Cys TCC Ser TTC Phe GGA Gly GCA Ala GTA Val GGC Gly GCC AlaGTC Val GGT Gly GCT Ala GTT Val GGG Gly GCG Ala GTG Val TGT Cys TCT SerTTT Phe GGG Gly GCG Ala GTG Val TGT Cys TCT Ser TTT Phe CGT Arg CCT ProCTT Leu AGT Ser ACT Thr ATT Ile CGG Arg CCG Pro CTG Leu TGT Cys TCT SerTTT Phe CGC Arg CCC Pro CTC Leu CGG Arg CCG Pro CTG Leu TGG Trp TCG SerTTG Leu CGG Arg CCG Pro CTG Leu TGT Cys TCT Ser TTT Phe AGT Ser ACT ThrATT Ile TGT Cys TCT Ser TTT Phe AGG Arg ACG Thr ATG Met TGG Trp TCG SerTTG Leu AGC Ser ACC Thr ATC Ile AGT Ser ACT Thr ATT Ile CGT Arg CCT ProCTT Leu AGT Ser ACT Thr ATT Ile CGC Arg CCC Pro CTC Leu GGT Gly GCT AlaGTT Val TGT Cys TCT Ser TTT Phe CGG Arg CCG Pro CTG Leu AGA Arg ACA ThrATA Ile CGT Arg CCT Pro CTT Leu AGC Ser ACC Thr ATC Ile CGT Arg CCT ProCTT Leu TGC Cys TCC Ser TTC Phe AGG Arg ACG Thr ATG Met GGT Gly GCT AlaGTT Val GGC Gly GCC Ala GTC Val Codon Amino Acid Codon Amino Acid CodonAmino Acid Codon Amino Acid AAA Lys AAG Lys AAC Asn AAT Asn AAA Lys AAGLys AAC Asn AAT Asn CCA Pro CCG Pro CCC Pro CCT Pro CAA Gln CAG Gln CACHis CAT His ACA Thr ACG Thr ACC Thr ACT Thr CCA Pro CCG Pro CCC Pro CCTPro AAA Lys AAG Lys AAC Asn AAT Asn TTA Leu TTG Leu TTC Phe TTT Phe GCAAla GCG Ala GCC Ala GCT Ala GTA Val GTG Val GTC Val GTT Val GAA Glu GAGGlu GAC Asp GAT Asp GTA Val GTG Val GTC Val GTT Val TCA Ser TCG Ser TCCSer TCT Ser GCA Ala GCG Ala GCC Ala GCT Ala TAA Stop TAG Stop TAC TyrTAT Tyr CAA Gln CAG Gln CAC His CAT His ATA Ile ATG Met ATC Ile ATT IleCCA Pro CCG Pro CCC Pro CCT Pro TTA Leu TTG Leu TTC Phe TTT Phe CTA LeuCTG Leu CTC Leu CTT Leu CTA Leu CTG Leu CTC Leu CTT Leu TTA Leu TTG LeuTTC Phe TTT Phe CAA Gln CAG Gln CAC His CAT His TTA Leu TTG Leu TTC PheTTT Phe ACA Thr ACG Thr ACC Thr ACT Thr TAA Stop TAG Stop TAC Tyr TATTyr ATA Ile ATG Met ATC Ile ATT Ile TTA Leu TTG Leu TTC Phe TTT Phe AAALys AAG Lys AAC Asn AAT Asn ACA Thr ACG Thr ACC Thr ACT Thr CCA Pro CCGPro CCC Pro CCT Pro AAA Lys AAG Lys AAC Asn AAT Asn CAA Gln CAG Gln CACHis CAT His GCA Ala GCG Ala GCC Ala GCT Ala TCA Ser TCG Ser TCC Ser TCTSer CAA Gln CAG Gln CAC His CAT His AAA Lys AAG Lys AAC Asn AAT Asn CTALeu CTG Leu CTC Leu CTT Leu AAA Lys AAG Lys AAC Asn AAT Asn CAA Gln CAGGln CAC His CAT His TTA Leu TTG Leu TTC Phe TTT Phe ATA Ile ATG Met ATCIle ATT Ile GGA Gly GGG Gly GGC Gly GGT Gly GTA Val GTG Val GTC Val GTTVal

TABLE 14 Amino acid mutations at target sites in rt Target Mutation K32M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L N33D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R P34S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F H35I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G T37W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S P59S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F K60M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L F61P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M A62R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V V63A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y D83C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N V84A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y S85T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P A86R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V Y89V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W H90I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G I/L91K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H P177S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F F178P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M L179K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I L180K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I A181R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V Q183E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C F183P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M T184W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S Y203V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W M204F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K L235K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I N236D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R T237W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S P237S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F N238D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R H238I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G A238R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V S239T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P Q238E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C K239M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L L247K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I N248D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V/A/R H248I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G F249P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K/M M250F/P/S/T/W/Y/V/A/R/N/D/C/Q/E/G/H/I/L/K G251H/I/L/K/M/F/P/S/T/W/Y/V/A/R/N/D/C/Q/E V251A/R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y

TABLE 15 Summary of HBV mutations in Patient F treated with ADV SampleName Genotype Polymerase* Surface CAP 01564808 A rtL157L/M sF83S rtA181VsL173F rtV207I sW199L rtN236T *Nomenclature according to Stuyver et al.,2001, supra. **Mutations in bold have not been detected in reference HBVgenotypes, mutations not in bold are changes from the previous samplethat are present in reference genotypes.

TABLE 16 Summary of HBV mutations in Patient G treated with ADV SampleName Genotype Polymerase* Surface KAN 02510355 C rtL80V sI126T rtP109SsK160R rtI163V sS174N rtM204I sA184V rtL229M sW196L rtN/H/A/S/Q238KsS210N sF/C220L sY221C *Nomenclature according to Stuyver et al., 2001,supra. **Mutations in bold have not been detected in reference HBVgenotypes, mutations not in bold are changes from the previous samplethat are present in reference genotypes.

TABLE 17 Summary of HBV mutations in Patient H treated with ADV SampleName Genotype Polymerase* Surface LAV0303 D rtS78S/T sC69Stop/CrtN118N/S sC76Y rtN139N/K sI110V/I rtV142E sY134N rtA181A/T sW172Stop/WrtI204M sW196Stop rtQ/P/S/Stop215Q sS207R rtE218K/E rtN238N/H*Nomenclature according to Stuyver et al., 2001, supra. **Mutations inbold have not been detected in reference HBV genotypes, mutations not inbold are changes from the previous sample that are present in referencegenotypes.

TABLE 18 In vitro drug susceptibility of the HBV reference laboratorystrain and patient-derived HBV isolate In vitro Susceptibility IC₅₀(fold change from wild-type) Real-time PCR Southern Blot MutationAdefovir Adefovir Lamivudine Wild-type (pPC) 1 1 1 rt N236T/N238D 23 NA¹NA¹ rt A181T/N236T/N238D 5.1 7.3 >100 rt L180M/M204V² NT⁵ 0.9 >2500 ¹NA,not analyzed. ²Data from Delaney et al., 2001, supra

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features

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The invention claimed is:
 1. A method for determining whether ahepatitis B virus (“test virus”) from a human patient would exhibitreduced sensitivity to a substituted acyclic nucleoside phosphideselected from adefovir and tenofovir, the method comprising: screening anucleic acid molecule from the test virus for an adefovir- ortenofovir-resistant co-mutation at the codon encoding amino acidposition 181 from alanine to threonine or valine and the codon encodingamino acid position 236 from asparagine to threonine where the aminoacid position is numbered with reference to the methionine in the motifYMDD being residue 204, wherein the nucleic acid molecule comprising anucleic acid sequence that encodes a reverse transcriptase domain of aDNA polymerase; and; wherein the mutation indicates that the test viruswould exhibit reduced sensitivity to the substituted acyclic nucleosidephosphate.
 2. The method of claim 1 wherein a hepatitis B virus with theco-mutation at the codon encoding amino acid position 181 from alanineto threonine or valine and the codon encoding amino acid position 236from asparagine to threonine further comprises a mutation at the codonencoding amino acid position 84 from valine to methionine.
 3. The methodof claim 1 wherein the hepatitis B virus comprises co-mutations at thecodon encoding amino acid position 181 from alanine to threonine and atthe codon encoding amino acid position 236 from asparagine to threonineand further comprises a mutation at the codon encoding amino acidposition 214 from valine to alanine.